Neublastin neurotrophic factors

ABSTRACT

The invention relates to neublastin neurotrophic factor polypeptides, nucleic acids encoding neublastin polypeptides, and antibodies that bind specifically to neublastin polypeptides, as well as methods of making and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/347,613 (now U.S. Pat. No. 6,593,133), filed Jul. 2, 1999,which claims the benefit of U.S. Serial No. 60/092,229, filed Jul. 9,1998; U.S. Serial No. 60/097,774, filed Aug. 25, 1998, and U.S. SerialNo. 60/103,908, filed Oct. 13, 1998 each hereby incorporated byreference in their entirety. This application also claims benefit ofDanish patent application 1998 00904, filed Jul. 6, 1998; Danish patentapplication 1998 01048, filed Aug. 19, 1998 and Danish patentapplication 1998 01265, filed Oct. 6, 1998.

FIELD OF THE INVENTION

The invention relates to neurotrophic factor polypeptides, nucleic acidsencoding neurotrophic factor polypeptides, and antibodies that bindspecifically to neurotrophic factors.

BACKGROUND

Neurotrophic factors are naturally-occurring proteins which promotesurvival, maintain phenotypic differentiation, prevent degeneration, andenhance the activity of neuronal cells and tissues. Neurotrophic factorsare isolated from neural tissue and from non-neural tissue that isinnervated by the nervous system, and have been classified intofunctionally and structurally related groups, also referred to asfamilies, superfamilies, or subfamilies. Among the neurotrophic factorsuperfamilies are the fibroblast growth factor, neurotrophin, andtransforming growth factor-β (TGF-β) superfamilies. Individual speciesof neurotrophic factors are distinguished by their physical structure,their interaction with their composite receptors, and their affects onvarious types of nerve cells. Classified within the TGF-β superfamily(Massague, et al,. 1994, Trends in Cell Biology, 4:172-178) are theglial cell line-derived neurotrophic factor ligands (“GDNF”; WO93/06116, incorporated herein by reference), which include GDNF,persephin (“PSP”; Milbrandt et al., 1998, Neuron 20:245-253,incorporated herein by reference) and neurturin (“NTN”; WO 97/08196,incorporated herein by reference). The ligands of the GDNF subfamilyhave in common their ability to induce signalling through the RETreceptor tyrosine kinase. These three ligands of the GDNF subfamilydiffer in their relative affinities for a family of neurotrophicreceptors, the GFRα receptors.

Due to the affects of neurotrophic factors on neuronal tissue, thereremains a need to identify and characterise additional neurotrophicfactors for diagnosing and treating disorders of the nervous system.

SUMMARY OF THE INVENTION

This invention relates to a novel neurotrophic factor herein called“neublastin,” or “NBN.” Neublastin is classified within the GDNFsubfamily because it shares regions of homology with other GDNF ligands(see Tables 3 and 4, infra) and because of its ability to interact withRET (see, e.g., Airaksinen et al., Mol. Cell. Neuroscience, 13, pp.313-325 (1999)), neublastin is a novel and unique neurotrophic factor.Unlike other GDNF ligands, neublastin exhibits high affinity for theGFRα3-RET receptor complex and unique subregions in its amino acidsequence.

A “neublastin polypeptide,” as used herein, is a polypeptide whichpossesses neurotrophic activity (e.g., as described in Examples 6, 7, 8,and 9) and includes those polypeptides which have an amino acid sequencethat has at least 70% homology to the human “neublastin” polypeptidesset forth in AA⁻⁹⁵-AA₁₀₅ of SEQ. ID. NO. 2, AA₁-AA₁₀₅ of SEQ. ID. NO. 2,AA⁻⁹⁷-AA₁₄₀ of SEQ. ID. NO. 4, AA⁻⁴¹-AA₁₄₀ of SEQ. ID. NO. 4 (“pro”),AA₁-AA₁₄₀ of SEQ. ID. NO. 4, AA⁻⁸⁰-AA₁₄₀ of SEQ. ID. NO. 9 (“wild type”prepro), AA⁻⁴¹-AA₁₄₀ of SEQ. ID. NO. 9 (pro), AA₁-AA₁₄₀ of SEQ. ID. NO.5 (mature 140AA), AA₁-AA₁₁₆ of SEQ. ID. NO. 6 (mature 116AA), AA₁-AA₁₁₃of SEQ. ID. NO. 7 (mature 113AA), AA₁-AA₁₄₀ of SEQ. ID. NO. 10 (mature140AA), AA₁-AA₁₁₆ of SEQ. ID. NO. 11 (mature 116AA), AA₁-AA₁₁₃ of SEQ.ID. NO. 12 (mature 113AA), and variants and derivatives thereof. Inaddition, this invention contemplates those polypeptides which have anamino acid sequence that has at least 70% homology to the murine“neublastin” polypeptides set forth in AA₁-AA₂₂₄ of SEQ. ID. NO. 16.

Preferably, the C-terminal sequence of the above identified neublastinpolypeptides has an amino acid sequence as set forth in AA₇₂-AA₁₀₅ ofSEQ. ID. NO. 2 (i.e., AA₁₀₇-AA₁₄₀ of SEQ. ID. NO. 9), more preferablyAA₄₁-AA₁₀₅ of SEQ. ID. NO. 2 (i.e., AA₇₆-AA₁₄₀ of SEQ. ID. NO. 9), orthe amino acid sequence set forth in AA₁₉₁-AA₂₂₄ of SEQ. ID. NO. 16.

Also, it is preferable that the neublastin polypeptide retain the 7conserved Cys residues that are characteristic of the GDNF family and ofthe TGF-beta super family.

Preferably, the neublastin polypeptide has an amino acid sequencegreater than 85% homology, most preferably greater than 95% homology, tothe foregoing sequences (i.e., AA⁻⁹⁵-AA₁₀₅ of SEQ. ID. NO. 2, AA₁-AA₁₀₅of SEQ. ID. NO. 2, AA⁻⁹⁷-AA₁₄₀ of SEQ. ID. NO. 4, AA₁-AA₁₄₀ of SEQ. ID.NO. 4, AA⁻⁴¹-AA₁₄₀ of SEQ. ID. NO. 4, AA⁻⁸⁰-AA₁₄₀ of SEQ. ID. NO. 9(“wild type” prepro), AA⁻⁴¹-AA₁₄₀ of SEQ. ID. NO. 9 (pro), AA₁-AA₁₄₀ ofSEQ. ID. NO. 5 (mature 140AA), AA₁-AA₁₁₆ of SEQ. ID. NO. 6 (mature116AA), AA₁-AA₁₁₃ of SEQ. ID. NO. 7 (mature 113AA), AA₁-AA₁₄₀ of SEQ.ID. NO. 10 (mature 140AA), AA₁-AA₁₁₆ of SEQ. ID. NO. 11 (mature 116AA),AA₁-AA₁₁₃ of SEQ. ID. NO. 12 (mature 113AA)), and AA₁-AA₂₂₄ of SEQ. ID.NO. 16.

A “neublastin nucleic acid,” as used herein, is a polynucleotide whichcodes for a neublastin polypeptide. Accordingly, an isolated neublastinnucleic acid is a polynucleotide molecule having an open reading frameof nucleotide codons that, were it to be exposed to the appropriatecomponents required for translation, would encode, or code for, aneublastin polypeptide. Neublastin nucleic acids of the invention may beRNA or DNA, e.g., genomic DNA, or DNA which is complementary to and/ortranscribed from, a neublastin mRNA (“cDNA”). Thus, a neublastin nucleicacid of the invention further includes polynucleotide molecules whichhybridize with specificity, under high stringency hybridizationconditions, to a polynucleotide that codes for a neublastin polypeptide.This invention also relates to nucleic acid primers that are useful inidentifying, isolating and amplifying polynucleotides that encodeneublastin polypeptides, or fragments thereof. In certain embodiments ofthe invention, certain of these primers are neublastin-specific probesuseful for hybridization to a neublastin nucleic acid, but not tonucleic acids coding for the other members of the GDNF family. By“specific”, “specificity”, or “specifically”, is meant an ability tohybridize with neublastin nucleic acid and inability to hybridize withnon-neublastin nucleic acids, including an inability to hybridize tonucleic acids that code uniquely for the GDNF ligands (e.g., GDNF,persephin, and neurturin).

In another embodiment, a neublastin nucleic acid of the invention is onethat is identified as being complementary to a polynucleotide that codesfor a neublastin polypeptide, either by having a complementary nucleicacid sequence or demonstrating that it hybridizes with specificity athigh stringency hybridization conditions to a polynucleotide that codesfor neublastin. Particular neublastin nucleic acids include, withoutlimitation, the nucleic acid sequences shown herein and designated SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 29 and SEQ ID NO: 30 as well as primers SEQ IDNOS: 17-28, 31 and 32. A neublastin nucleic acid of the inventionfurther includes a unique subregion, or fragment, of a neublastinnucleic acid, including without limitation the nucleic acid fragmentsshown in FIG. 8.

The neublastin nucleic acids of the invention may be used to express aneublastin polypeptide, e.g., by expressing a neublastin polypeptide invitro, or by administering a neublastin nucleic acid to an animal for invivo expression. Neublastin nucleic acids may be included within anucleic acid vector, e.g., an expression vector or a cloning vector. Aneublastin nucleic acid may, but need not of necessity, be maintained,reproduced, transferred, or expressed as part of a nucleic acid vector.A recombinant expression vector containing a neublastin polynucleotidesequence can be introduced into and/or maintained within a cell. Cellshosting a neublastin vector may be prokaryotic. Alternatively, aneublastin nucleic acid can be introduced into a eukaryotic cell, e.g.,a eukaryotic cell that contains the appropriate apparati forpost-translational processing of a polypeptide into a mature protein,and/or the appropriate apparati for secreting a polypeptide into theextracellular environment of the cell.

The invention further features a neublastin neurotrophic factor,“neublastin.” Neublastin may be in the form of a polypeptide, or may bea multimer of two or more neublastin polypeptides, e.g., a neublastindimer. Neublastin polypeptides are associated as multimers byintermolecular structural associations known to those skilled in theart, including without limitation cysteine-cysteine interaction,sulfhydryl bonds, and noncovalent interactions. Particular neublastinpolypeptides include, without limitation, an amino acid sequencedisclosed herein and designated SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO:5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO:11, SEQ ID NO: 12 and SEQ ID NO: 16.

A neublastin polypeptide of the invention is useful for treating adefect in a neuron, including without limitation lesioned neurons andtraumatized neurons. Peripheral nerves that experience trauma include,but are not limited to, nerves of the medulla or of the spinal cord.Neublastin polypeptides are useful in the treatment of neurodegenerativedisease, e.g., cerebral ischemic neuronal damage; neuropathy, e.g.,peripheral neuropathy, Alzheimer's disease, Huntington's disease,Parkinson's disease, amyotrophic lateral sclerosis (ALS). Neublastinpolypeptides are further contemplated for use in the treatment ofimpaired memory, e.g., memory impairment associated with dementia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photographic image of two northern blots probed with³²P-labelled neublastin cDNA, comparing relative levels of expression ofthe neublastin gene in various human adult tissue types (panel A) and invarious regions of the adult human brain (panel B).

FIG. 2 is a photographic image of a northern blot probed with32P-labelled neublastin cDNA, comparing the amount of neublastin cDNAexpressed in a non-transfected cell-line, HiB5, with the amount ofneublastin cDNA expressed in a cell-line transfected with neublastincDNA, and with a cell-line transfected with GDNF-cDNA.

FIG. 3 is a photographic image of two western blots which compare thedegrees to which neublastin protein is expressed in non-transfected HiB5cells (lane 1) relative to an HiB5 cell-line stably-transfected withneublastin cDNA (lane 2) was probed with either the neublastin-specificantibody Ab-2 (left blot; Panel A) or the neublastin-specific antibodyAb-1 (right blot; Panel B).

FIG. 4 is a graphical illustration of the effect of neublastin on thesurvival of cultured rat embryonic, dopaminergic, ventral mesencephalicneurons and ChAT activity in cholinergic cranial nerve motor neurons inserum-free medium. In particular, FIG. 4A is an illustration of thedose-response curve for recombinant GDNF on ChAT activity (dpm/hour).FIG. 4B is an illustration of ChAT activity (dpm/hour) using dilutedconditioned medium from either neublastin producing or GDNF-producingcells. FIG. 4C is an illustration of the number of tyrosine hydroxylaseimmunoreactive cells per well.

FIG. 5 is an illustration of the effect of neublastin secreted fromHiB5pUbi1zNBN22 cells on the function and survival of slice cultures ofpig embryonic dopaminergic ventral mesencephalic neurons co-culturedwith either HiB5pUbi1zNBN22 cells (neublastin) or HiB5 cells (control).FIG. 5A and FIG. 5B illustrate dopamine released to the medium at DIV12[Dopamine (pmol/ml)−day 12] and DIV21 [Dopamine (pmol/ml)−day 21],respectively. FIG. 5C is an illustration of the number of tyrosinehydroxylase immunoreactive cells per culture [TH-ir cells per culture]at DIV21.

FIG. 6 is an illustration of the in vivo effect of lentiviral-producedneublastin on nigral dopamine neurons.

FIG. 7 is a schematic diagram of the genomic structure of the neublastingene, including the nucleic acid primers which can be used to identifythe full length neublastin gene, and their spatial orientation inrelation to the genomic Neublastin-encoding sequence (i.e., gene).

FIG. 8 is an illustration of neublastin specific primers used toidentify the cDNA clone encoding the human neublastin polypeptide thathybridize to nucleic acids that encode neublastin polypeptides, but donot hybridize to nucleic acids encoding the other known GDNF familymembers (i.e., GDNF (SEQ ID NOS: 40 and 43), Persephin (SEQ ID NOS: 38and 41) and neurturin (SEQ ID NOS: 39 and 42)).

FIG. 9 illustrates the neurotrophic activity on cultures of dissociatedrat dorsal root ganglion cells from different development stages of apolypeptide disclosed in the present invention in comparison to thoseobtained with known neurotrophic factors [0 control experiment (inabsence of factors); 1 in the presence of GDNF; 2 in the presence ofNeurturin; 3 in the presence of Neublastin of the invention; E12embryonic day 12; E16 embryonic day 16; P0 the day of birth; P7 day 7after birth; and P15 day 15 after birth].

FIG. 10 illustrates neublastin production from CHO cell lines.

FIG. 11 illustrates a comparison of neublastin and GDNF binding toGFRα-1 and GFRα-3 receptors.

FIG. 12 is a photographic image of a western blot which illustrates R30anti-peptide antibody and R31 anti-peptide antibody binding toneublastin.

FIG. 13 is a picture of a gel showing extraction of neublastin byaffinity binding on RETL3-Ig.

FIG. 14 is a plasmid map of pET19b-Neublastin, along with the sequenceof the synthetic gene for Neublastin.

FIG. 15 is a plasmid map of pMJB164-HisNeublastin, along with thesequence of the synthetic gene for HisNeublastin.

DETAILED DISCLOSURE OF THE INVENTION

Applicant have identified a nucleic acid that encodes a novelneurotrophic factor which is referred to herein as “neublastin,” or“NBN.” Neublastin is a member of the glial cell line-derivedneurotrophic factor (GDNF) sub-class of the transforming growth factor-β(TGF-β) super-family of neurotrophic factors.

The cDNA encoding neublastin was originally identified as follows. Usingthe TBLASTN 1.4.11 algorithm (Atschul et al., Nucl. Acids Res., 25, pp.3389-3402 (1997)) and human persephin as query (GenBank Acc. No.AF040962), a 290 bp fragment was initially identified in High-ThroughputGenomic Sequence (HGTS) of two human bacterial artificial chromosomes(BAC) with GenBank entries AC005038 and AC005051. AC005038 consists ofapproximately 190,000 bp of 5 contigs of unordered sequences andAC005051 consists of approximately 132,000 bp of 12 contigs of unorderedsequences. The 290 bp fragment identified in the two BAC clones provedto have regions that were homologous, but not identical, to a codingregion of the cDNA of the neurotrophic factor, human persephin.

From this 290 bp sequence two Neublastin-specific PCR primers weresynthesised (Top Stand Primer [SEQ ID NO. 17] and Bottom Strand Primer[SEQ ID NO. 18]). Screening of human fetal brain cDNA library wasperformed. The initial screening comprised 96-well PCR-based screeningwith the two PCR primers [SEQ ID NOS. 17 and 18] of a cDNA library“Master Plate” from 500,000 cDNA clones containing approximately 5,000clones/well. A second PCR-based screen was performed on a human fetalbrain cDNA library “Sub-Plate” containing E. coli glycerol stock withapproximately 5,000 clones/well.

A 102 bp fragment [SEQ ID NO. 13] was identified in the PCR-basedscreenings of both the Master Plate and Sub Plate. A positive cDNA clone(possessing the 102 bp fragment) was selected, plated on twoLB/antibiotic-containing plates, and grown overnight. From these plates,a total of 96 bacterial colonies were selected and individually placedin the wells of a new, 96-well PCR plate containing both PCR primers[SEQ ID NOS. 17 and 18] and the requisite PCR amplification reagents.PCR amplification was then performed and the 96 individual PCR reactionswere analyzed by 2% agarose gel electrophoresis. The positive colonywith the clone containing the 102 bp fragment was then identified.Plasmid DNA was obtained from the positive colony containing the 102 bpfragment and sequenced. Subsequent sequencing analysis revealed thepresence of a full-length cDNA of 861 bp [SEQ ID NO. 8]. The OpenReading Frame (ORF) of 663 bp, or coding region (CDS), identified in SEQID NO. 8, encodes the pre-pro-polypeptide (designated“pre-pro-Neublastin”) and is shown in SEQ ID NO: 9. Based on SEQ ID NO:9, three variants of Neublastin polypeptides were identified. Thesevariants include:

(i) the 140 AA polypeptide designated herein as NBN140, which possessesthe amino acid sequence designated as SEQ ID NO: 10;

(ii) the 116 AA polypeptide designated herein as NBN116, which possessesthe amino acid sequence designated as SEQ ID NO: 11; and

(iii) the 113 AA polypeptide designated herein as NBN113, whichpossesses the amino acid sequence designated as SEQ ID NO: 12.

The entire cDNA sequence containing 782 bp 5′ untranslated DNA, 663 bpencoding DNA, and 447 3′ untranslated (totalling 1992 bp) has beensubmitted to GenBank under the Accession Number AF 120274.

The genomic Neublastin-encoding sequence was identified as follows:

With the goal of cloning the genomic neublastin-encoding sequence, anadditional set of primers were prepared. In particular, Primer Pair No.1 comprised [sense=SEQ ID NO:23 and antisense=SEQ ID NO:24] and PrimerPair No. 2 comprised [sense=SEQ ID NO:25 and antisense=SEQ ID NO:26].

Using Primer Pair No. 2, a 887 bp DNA fragment was amplified by PCR froma preparation of human genomic DNA, and cloned into the pCRII vector(Invitrogen) and transformed into E. coli. The resulting plasmid wassequenced and a 861 bp putative cDNA sequence (encoding a protein namedneublastin herein) was predicted (as set forth in SEQ.ID.NO.3).Similarly, using Primer Pair No. 1, an 870 bp DNA fragment was obtainedby PCR of human genomic DNA. An additional 42 bp region at the3′-terminus of the Open Reading Frame (ORF) was found in this fragment,in comparison to the 887 bp sequence. The genomic structure of theneublastin gene was predicted by comparing it to the sequences ofnucleic acids of other neurotrophic factors, by mapping exon-intronboundaries. This analysis demonstrated that the neublastin gene has atleast two exons separated by a 70 bp intron.

This sequence was also used to screen GenBank for neublastin ESTsequences. Three were identified with GenBank entries AA844072, AA931637and AA533512, indicating that neublastin nucleic acids are transcribedinto mRNA.

Comparison of the entire cDNA sequence obtained (AF 120274) and thegenomic sequence present in GenBank entries AC005038 and AC005051revealed that the neublastin gene consists of at least five exons(including three coding) separated by four introns (see, e.g., FIG. 8).Together, the exons have a predicted amino acid sequence of afull-length Neublastin polypeptide. It should also be noted that the 887bp fragment was found to contain the complete coding region ofpro-neublastin. The predicted cDNA [SEQ ID NO: 3] contains an OpenReading Frame (ORF) encoding pro-neublastin (181 amino acid residues)which showed homology to the three known human proteins—Persephin,Neurturin, and GDNF.

Neublastin Nucleic Acids of the Invention

In another aspect the invention provides polynucleotides capable ofexpressing the polypeptides of the invention. The polynucleotides of theinvention include DNA, cDNA and RNA sequences, as well as anti-sensesequences, and include naturally occurring, synthetic, and intentionallymanipulated polynucleotides. The polynucleotides of the invention alsoinclude sequences that are degenerate as a result of the genetic code,but which code on expression for a neublastin polypeptide.

As defined herein, the term “polynucleotide” refers to a polymeric formof nucleotides of at least 10 bases in length, preferably at least 15bases in length. By “isolated polynucleotide” is meant a polynucleotidethat is not immediately contiguous with both of the coding sequenceswith which it is immediately contiguous (one on the 5′ end and one onthe 3′ end) in the naturally occurring genome of the organism from whichit is derived. The term therefore includes recombinant DNA which isincorporated into an expression vector, into an autonomously replicatingplasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule, e.g. a cDNA, independent fromother sequences.

The polynucleotides of the invention also include allelic variants and“mutated polynucleotides” having a nucleotide sequence that differs fromthe nucleotide sequences presented herein at one or more nucleotidepositions.

In a preferred embodiment, the polynucleotide of the invention has anucleic acid (DNA) sequence capable of hybridizing with thepolynucleotide sequence presented as SEQ ID NO: 1, the polynucleotidesequence presented as SEQ ID NO: 3, the polynucleotide sequencepresented as SEQ ID NO: 8, or the polynucleotide sequence presented asSEQ ID NO: 15, its complementary strand, or a sub-sequence hereof underat least medium, medium/high, or high stringency conditions, asdescribed in more detail below.

In another preferred embodiment, the isolated polynucleotide of theinvention has a nucleic acid (DNA) sequence that is at least 70%,preferably at least 80%, more preferred at least 90%, most preferred atleast 95% homologous to the polynucleotide sequence presented as SEQ IDNO: 1, the polynucleotide sequence presented as SEQ ID NO: 3, thepolynucleotide sequence presented as SEQ ID NO: 8, or the polynucleotidesequence presented as SEQ ID NO: 15.

In its most preferred embodiment, the polynucleotide has the DNAsequence presented as SEQ ID NO: 1, the DNA sequence presented as SEQ IDNO: 3, the DNA sequence presented as SEQ ID NO: 8, or the polynucleotidesequence presented as SEQ ID NO: 15.

This invention also provides novel primers and DNA sequences foridentifying, isolating and amplifying neublastin polynucleotides whichcode on expression for neublastin polypeptides or fragments thereof.Such primers include the polynucleotides set forth in SEQ.ID.NOS. 17-28,and 31-32. In addition, this invention provides neublastin DNA sequencesgenerated from those primers, including those set forth in SEQ.ID.NOS.13 and 14. Further, this invention provides DNA sequences from 3′ or 5′untranslated regions (“UTR”) in genomic DNA that flank neublastin exons;such sequences are useful in identifying, isolating and amplifyingneublastin polynucleotides which code on expression for neublastinpolypeptides or fragments thereof

3′ UTR sequences of this invention include the sequences set forth in:

nucleotides 721-865 of SEQ.ID.NO. 1,

nucleotides 718-861 of SEQ.ID.NO. 3,

nucleotides 718-861 of SEQ.ID.NO. 8,

nucleotides 1647-2136 of SEQ.ID.NO. 15, and

contiguous sequences of between 10-25 nucleotides derived from (i.e.,falling within) the foregoing sequences (which are useful, e.g., asprimers).

5′ UTR sequences of this invention include the sequences set forth in:

nucleotides 1-10 of SEQ.ID.NO. 1,

nucleotides 1-57 of SEQ.ID.NO. 8,

nucleotides 1-974 of SEQ.ID.NO. 15, and

contiguous sequences of between 10-25 nucleotides derived from (i.e.,falling within) the foregoing sequences (which are useful, e.g., asprimers).

The polynucleotides of the invention may preferably be obtained bycloning procedures, e.g. as described in “Current Protocols in MolecularBiology” [John Wiley & Sons, Inc.]. In a preferred embodiment, thepolynucleotide is cloned from, or produced on the basis of human genomicDNA or a cDNA library of the human brain.

Homology of DNA Sequences

The DNA sequence homology referred to above may be determined as thedegree of identity between two sequences indicating a derivation of thefirst sequence from the second. The homology may suitably be determinedby means of computer programs known in the art, such as GAP provided inthe GCG program package [Needleman, S. B. and Wunsch C. D., Journal ofMolecular Biology 1970 48 443-453]. Using GAP with the followingsettings for DNA sequence comparison: GAP creation penalty of 5.0 andGAP extension penalty of 0.3, the coding region of the analogous DNAsequences referred to above exhibits a degree of identity preferably ofat least 70%, more preferably at least 80%, more preferably at least90%, more preferably at least 95%, with the CDS (encoding) part of theDNA sequence shown in SEQ ID No. 1, or the CDS (encoding) part of theDNA sequence shown in SEQ ID No. 3, or the CDS (encoding) part of theDNA sequence shown in SEQ ID No. 8, the CDS (encoding) part of the DNAsequence shown in SEQ.ID.NO. 15.

The term “sequence identity” refers to the degree to which twopolynucleotide sequences are identical on a nucleotide-by-nucleotidebasis over a particular region of comparison. The term “percentage ofsequence identity” is calculated by comparing two optimally alignedsequences over that region of comparison, determining the number ofpositions at which the identical nucleic acid base (e.g., A, T, C, G, U,or I) occurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the region of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity. The term “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 80 percent sequence identity,preferably at least 85 percent identity and often 90 to 95 percentsequence identity, more usually at least 99 percent sequence identity ascompared to a reference sequence over a comparison region.

Hybridization Protocol

The polynucleotides of the invention are such which have a nucleic acidsequence capable of hybridizing with the polynucleotide sequencepresented as SEQ ID NO: 1, the polynucleotide sequence presented as SEQID NO: 3, or the polynucleotide sequence presented as SEQ ID NO: 8, orthe polynucleotide sequence presented as SEQ ID NO: 15, or theircomplementary strand, or a sub-sequence hereof under at least medium,medium/high, or high stringency conditions, as described in more detailbelow.

Suitable experimental conditions for determining hybridization between anucleotide probe and a homologous DNA or RNA sequence, involvespre-soaking of the filter containing the DNA fragments or RNA tohybridize in 5×SSC [Sodium chloride/Sodium citrate; cf. Sambrook et al.;Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., ColdSpring Harbor, N.Y. 1989] for 10 minutes, and pre-hybridization of thefilter in a solution of 5×SSC, 5×Denhardt's solution [cf. Sambrook etal.; Op cit.], 0.5% SDS and 100 μg/ml of denatured sonicated salmonsperm DNA [cf. Sambrook et al.; Op cit.], followed by hybridization inthe same solution containing a concentration of 10 ng/ml of arandom-primed [Feinberg A P & Vogelstein B; Anal. Biochem. 1983 1326-13], ³²P-dCTP-labeled (specific activity>1×10⁹ cpm/μg) probe for 12hours at approximately 45° C. The filter is then washed twice for 30minutes in 0.1×SSC, 0.5% SDS at a temperature of at least at least 60°C. (medium stringency conditions), preferably of at least 65° C.(medium/high stringency conditions), more preferred of at least 70° C.(high stringency conditions), and even more preferred of at least 75° C.(very high stringency conditions). Molecules to which theoligonucleotide probe hybridizes under these conditions may be detectedusing a x-ray film.

Cloned Polynucleotides

The isolated polynucleotide of the invention may in particular be acloned polynucleotide. As defined herein, the term “clonedpolynucleotide”, refers to a polynucleotide or DNA sequence cloned inaccordance with standard cloning procedures currently used in geneticengineering to relocate a segment of DNA, which may in particular becDNA, i.e. enzymatically derived from RNA, from its natural location toa different site where it will be reproduced.

Cloning may be accomplished by any suitable route and may involvetechniques such as reverse transcriptase technology, PCR technology, andthe like, as well as excision and isolation of the desired DNA segment.

The cloned polynucleotide of the invention may alternatively be termed“DNA construct” or “isolated DNA sequence”, and may in particular be acomplementary DNA (cDNA).

Biological Sources

The isolated polynucleotide of the invention may be obtained from anysuitable source.

In a preferred embodiment, which the polynucleotide of the invention iscloned from, or produced on the basis of a cDNA library, e.g. of a cDNAlibrary of the fetal or adult brain, in particular of the forebrain, thehindbrain, the cortex, the striatum, the amygdala, the cerebellum, thecaudate nucleus, the corpus callosum, the hippocampus, the thalamicnucleus, the subthalamic nucleus, the olfactory nucleus, the putamen,the substantia nigra, the dorsal root ganglia, the trigeminal ganglion,the superior mesenteric artery, or the thalamus; of the spinal cord; ofthe heart; the placenta; of the lung; of the liver; of the skeletalmuscle; of the kidney; of the liver; of the pancreas; of the intestines;of the eye; of the retina; of the tooth pulp; of the hair follicle; ofthe prostate; of the pituitary; or of the trachea.

Commercial cDNA libraries from a variety of tissues, both human andnon-human, are available from e.g. Stratagene and Clontech. The isolatedpolynucleotide of the invention may be obtained by standard methods,e.g. those described in the working examples.

Neublastin Polypeptides of the Invention

As noted above, a “neublastin polypeptide,” as used herein, is apolypeptide which possesses neurotrophic activity (e.g., as described inExamples 6, 7, 8, and 9) and includes those polypeptides which have anamino acid sequence that has at least 70% homology to the “neublastin”polypeptides set forth in AA⁻⁹⁵-AA₁₀₅ of SEQ. ID. NO. 2, AA₁-AA₁₀₅ ofSEQ. ID. NO. 2, AA⁻⁹⁷-AA₁₄₀ of SEQ. ID. NO. 4, AA⁻⁴¹-AA₁₄₀ of SEQ. ID.NO. 4, AA₁-AA₁₄₀ of SEQ. ID. NO. 4, AA⁻⁸⁰-AA₁₄₀ of SEQ. ID. NO. 9 (“wildtype” prepro), AA⁻⁴¹-AA₁₄₀ of SEQ. ID. NO. 9 (pro), AA₁-AA₁₄₀ of SEQ.ID. NO. 5 (mature 140AA), AA₁-AA₁₁₆ of SEQ. ID. NO. 6 (mature 116AA),AA₁-AA₁₁₃ of SEQ. ID. NO. 7 (mature 113AA), AA₁-AA₁₄₀ of SEQ. ID. NO. 10(mature 140AA), AA₁-AA₁₁₆ of SEQ. ID. NO. 11 (mature 116AA), AA₁-AA₁₁₃of SEQ. ID. NO. 12 (mature 113AA), AA₁-AA₂₂₄ of SEQ. ID. NO. 16 (murineprepro), and variants and derivatives of each of the foregoing.

Preferably, the C-terminal sequence of the above identified neublastinpolypeptides has an amino acid sequence as set forth in AA₇₂-AA₁₀₅ ofSEQ. ID. NO. 2 (i.e., AA₁₀₇-AA₁₄₀ of SEQ. ID. NO. 9), more preferablyAA₄₁-AA₁₀₅ of SEQ. ID. NO. 2 (i.e., AA₇₆-AA₁₄₀ of SEQ. ID. NO. 9).

Also, it is preferable that the neublastin polypeptide retain the 7conserved Cys residues that are characteristic of the GDNF family and ofthe TGF-beta super family.

Preferably the neublastin polypeptide has an amino acid sequence greaterthan 85% homology, most preferably greater than 95% homology, to theforegoing sequences (i.e., AA⁻⁹⁵-AA₁₀₅ of SEQ. ID. NO. 2, AA₁-AA₁₀₅ ofSEQ. ID. NO. 2, AA⁻⁹⁷-AA₁₄₀ of SEQ. ID. NO. 4, AA⁻⁴¹-AA₁₄₀ of SEQ. ID.NO. 4, AA₁-AA₁₄₀ of SEQ. ID. NO. 4, AA⁻⁸⁰-AA₁₄₀ of SEQ. ID. NO. 9 (“wildtype” prepro), AA⁻⁴¹-AA₁₄₀ of SEQ. ID. NO. 9 (pro), AA₁-AA₁₄₀ of SEQ.ID. NO. 5 (mature 140AA), AA₁-AA₁₁₆ of SEQ. ID. NO. 6 (mature 116AA),AA₁-AA₁₁₃ of SEQ. ID. NO. 7 (mature 113AA), AA₁-AA₁₄₀ of SEQ. ID. NO. 10(mature 140AA), AA₁-AA₁₁₆ of SEQ. ID. NO. 11 (mature 116AA), AA₁-AA₁₁₃of SEQ. ID. NO. 12 (mature 113AA), AA₁-AA₂₂₄ of SEQ. ID. NO. 16 (murineprepro), and preferably any of the foregoing polypeptides with aC-terminal sequence of the above identified neublastin polypeptides hasan amino acid sequence as set forth in AA₇₂-AA₁₀₅ of SEQ. ID. NO. 2(i.e., AA₁₀₇-AA₁₄₀ of SEQ. ID. NO. 9), more preferably AA₄₁-AA₁₀₅ ofSEQ. ID. NO. 2 (i.e., AA₇₆-AA₁₄₀ of SEQ. ID. NO. 9) or AA₁₉₁-AA₂₂₄ ofSEQ. ID. NO. 16.

In addition, this invention contemplates those polypeptides which havean amino acid sequence that has at least 70% homology to the murine“neublastin” polypeptides set forth in AA₁-AA₂₂₄ of SEQ. ID. NO. 16.

Among the preferred polypeptides of the invention in one embodimentrepresent the preprosequence(as set forth in SEQ. ID. NOS. 2, 4, 9, and16, respectively), the pro sequence (as set forth in AA⁻⁷⁵-AA₁₀₅ of SEQ.ID. NO. 2, or AA⁻⁴¹-AA₁₄₀ of SEQ.ID.NOS. 4 and 9, respectively) and themature sequence of neublastin (as set forth in SEQ. ID. NOS. 5, 6, 7,10, 11, or 12, preferably SEQ. ID. NOS. 10, 11, 12).

The polypeptides of the invention include variant polypeptides. In thecontext of this invention, the term “variant polypeptide” means apolypeptide (or protein) having an amino acid sequence that differs fromthe sequence presented as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, or SEQ ID NO: 16, at one or more amino acid positions. Suchvariant polypeptides include the modified polypeptides described above,as well as conservative substitutions, splice variants, isoforms,homologues from other species, and polymorphisms.

As defined herein, the term “conservative substitutions” denotes thereplacement of an amino acid residue by another, biologically similarresidue. For example, one would expect conservative amino acidsubstitutions to have little or no effect on the biological activity,particularly if they represent less than 10% of the total number ofresidues in the polypeptide or protein. Preferably, conservative aminoacids substitutions represent changes in less than 5% of the polypeptideor protein, most preferably less than 2% of the polypeptide or protein(e.g., when calculated in accordance with NBN113, most preferredconservative substitutions would represent fewer than 3 amino acidsubstitutions in the wild type mature amino acid sequence). In aparticularly preferred embodiment, there is a single amino acidsubstitution in the mature sequence, wherein the both the substitutedand replacement amino acid are non-cyclic.

Other examples of particularly conservative substitutions include thesubstitution of one hydrophobic residue such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acid, or glutamine for asparagine, and the like.

The term conservative substitution also include the use of a substitutedamino acid residue in place of an un-substituted parent amino acidresidue provided that antibodies raised to the substituted polypeptidealso immunoreact with the un-substituted polypeptide.

Modifications of this primary amino acid sequence may result in proteinswhich have substantially equivalent activity as compared to theunmodified counterpart polypeptide, and thus may be consideredfunctional analogous of the parent proteins. Such modifications may bedeliberate, e.g. as by site-directed mutagenesis, or they may occurspontaneous, and include splice variants, isoforms, homologues fromother species, and polymorphisms. Such functional analogous are alsocontemplated according to the invention.

Moreover, modifications of the primary amino acid sequence may result inproteins which do not retain the biological activity of the parentprotein, including dominant negative forms, etc. A dominant negativeprotein may interfere with the wild-type protein by binding to, orotherwise sequestering regulating agents, such as upstream or downstreamcomponents, that normally interact functionally with the polypeptide.Such dominant negative forms are also contemplated according to theinvention.

A “signal peptide” is a peptide sequence that directs a newlysynthesized polypeptide to which the signal peptide is attached to theendoplasmic reticulum (ER) for further post-translational processing anddistribution.

An “heterologous signal peptide,” as used herein in the context ofneublastin, means a signal peptide that is not the human neublastinsignal peptide, typically the signal peptide of some mammalian proteinother than neublastin.

Skilled artisans will recognize that the human neublastin DNA sequence(either cDNA or genomic DNA), or sequences that differ from humanneublastin DNA due to either silent codon changes or to codon changesthat produce conservative amino acid substitutions, can be used togenetically modify cultured human cells so that they will overexpressand secrete the enzyme.

Polypeptides of the present invention also include chimeric polypeptidesor cleavable fusion polypeptides in which another polypeptide is fusedat the N-terminus or the C-terminus of the polypeptide or fragmentthereof. A chimeric polypeptide may be produced by fusing a nucleic acidsequence (or a portion thereof) encoding another polypeptide to anucleic acid sequence (or a portion thereof) of the present invention.

Techniques for producing chimeric polypeptides are standard techniques.Such techniques usually requires joining the sequences in a way so thatthey are in both in the same reading frame, and expression of the fusedpolypeptide under the control of the same promoter(s) and terminator.

Polypeptides of the present invention also include truncated forms ofthe full length neublastin molecule. In such truncated molecules, one ormore amino acids have been deleted from the N-terminus or theC-terminus, preferably the N-terminus.

Amino Acid Sequence Homology

The degree to which a candidate polypeptide shares homology with aneublastin polypeptide of the invention is determined as the degree ofidentity between two amino acid sequences. A high level of sequenceidentity indicates a likelihood that the first sequence is derived fromthe second

Homology is determined by computer analysis, such as, withoutlimitations, the ClustalX computer alignment program [Thompson J D,Gibson T J, Plewniak F, Jeanmougin F, & Higgins D G: The ClustalXwindows interface: flexible strategies for multiple sequence alignmentaided by quality analysis tools; Nucleic Acids Res. 1997, 25 (24):4876-82], and the default parameters suggested herein. Using thisprogram, the mature part of a polypeptide encoded by an analogous DNAsequence of the invention exhibits a degree of identity of at least 90%,more preferred of at least 95%, most preferred of at least 98% with theamino acid sequence presented herein as SEQ ID NO: 2, SEQ. ID. NO: 4;SEQ. ID. NO.: 5; SEQ. ID. NO.: 6; SEQ. ID. NO.: 7; SEQ. ID. NO.: 9; SEQ.ID. NO.: 10; SEQ. ID. NO.: 11; SEQ. ID. NO.: 12, or SEQ. ID. NO.: 16.

Based on the homology determination it is confirmed that the polypeptideof the invention, belonging to the TGF-β superfamily, is related to theGDNF subfamily, but represents a distinct member of this subfamily.

Bioactive Polypeptides

The polypeptide of the invention may be provided on any bioactive form,including the form of pre-pro-proteins, pro-proteins, mature proteins,glycosylated proteins, phosphorylated proteins, or any otherposttranslational modified protein.

The polypeptide of the invention may in particular be a N-glycosylatedpolypeptide, which polypeptide preferably is glycosylated at theN-residues indicated in the sequence listings.

In a preferred embodiment, the polypeptide of the invention has theamino acid sequence presented as SEQ ID NO: 9, holding a glycosylatedasparagine residue at position 122; the amino acid sequence presented asSEQ ID NO: 10, holding a glycosylated asparagine residue at position122; the amino acid sequence presented as SEQ ID NO: 11, holding aglycosylated asparagine residue at position 98; or the amino acidsequence presented as SEQ ID NO: 12, holding a glycosylated asparagineresidue at position 95.

This invention also contemplates neublastin fusion proteins, such asIg-fusions, as described, e.g., in U.S. Pat. No. 5,434,131, hereinincorporated by reference.

In one embodiment, the invention provides a polypeptide having the aminoacid sequence shown as SEQ ID NO: 2, or an amino acid sequence which isat least about 85%, preferably at least about 90%, more preferably atleast about 98%, and most preferably at least about 99% homologous tothe sequence presented as SEQ ID NO: 2.

In another embodiment, the invention provides a polypeptide having theamino acid sequence of SEQ ID NO: 4, or an amino acid sequence which isat least 90%, more preferred at least 95%, yet more preferred at least98%, most preferred at least 99% homologous to the sequence presented asSEQ ID NO: 4.

In a third embodiment, the invention provides a polypeptide having theamino acid sequence of SEQ ID NO: 5, or an amino acid sequence which isat least 90%, more preferred at least 95%, most preferred at least 98%homologous to the sequence presented as SEQ ID NO: 5.

In a fourth embodiment, the invention provides a polypeptides having theamino acid sequence of SEQ ID NO: 6, or an amino acid sequence which isat least 90%, more preferred at least 95%, most preferred at least 98%homologous to the sequence presented as SEQ ID NO: 6.

In a fifth embodiment, the invention provides a polypeptides having theamino acid sequence of SEQ ID NO: 7, or an amino acid sequence which isat least 90%, more preferred at least 95%, most preferred at least 98%homologous to the sequence presented as SEQ ID NO: 7.

The neublastin polypeptide of the invention includes allelic variants,e.g., the polypeptide amino acid sequences of SEQ ID NOS. 5-7, in whichXaa designates Asn or Thr, and Yaa designates Ala or Pro.

In a sixth embodiment, the invention provides a polypeptides having theamino acid sequence of SEQ ID NO: 9, or an amino acid sequence which isat least 90%, more preferred at least 95%, most preferred at least 98%homologous to the sequence presented as SEQ ID NO: 9.

In a seventh embodiment, the invention provides a polypeptide having theamino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least90%, more preferred at least 95%, most preferred at least 98%,homologous to the sequence presented as SEQ ID NO: 10.

In a eight embodiment, the invention provides a polypeptide having theamino acid sequence of SEQ ID NO: 11, or an amino acid sequence at least90%, more preferred at least 95%, most preferred at least 98% homologousto the sequence presented as SEQ ID NO: 11.

In a ninth embodiment, the invention provides a polypeptide having theamino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least90%, more preferred at least 95%, most preferred at least 98% homologousto the sequence presented as SEQ ID NO: 12.

In a tenth embodiment, the invention provides a polypeptide having theamino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least90%, more preferred at least 95%, most preferred at least 98% homologousto the sequence presented as SEQ ID NO: 16, which is apre-pro-neublastin of murine origin.

In another embodiment, the polypeptide of the invention holds the GDNFsubfamily fingerprint, i.e. the amino acid residues underlined in Table3.

In a further embodiment, the invention provides a polypeptide encoded bya polynucleotide sequence capable of hybridizing under high stringencyconditions with the polynucleotide sequence presented as SEQ ID NO: 1,its complementary strand, or a sub-sequence thereof. In a preferredembodiment, the polypeptide of the invention is encoded by apolynucleotide sequence being at least 70% homologous to thepolynucleotide sequence presented as SEQ ID NO: 1. In its most preferredembodiment, the polypeptide of the invention is encoded by thepolynucleotide sequence presented as SEQ ID NO: 1.

In a yet further embodiment, the invention provides novel polypeptidesencoded by a polynucleotide sequence capable of hybridizing under highstringency conditions with the polynucleotide sequence presented as SEQID NO: 3, its complementary strand, or a sub-sequence thereof. In apreferred embodiment, the polypeptide of the invention is encoded by apolynucleotide sequence being at least 70% homologous to thepolynucleotide sequence presented as SEQ ID NO: 3. In its most preferredembodiment, the polypeptide of the invention is encoded by thepolynucleotide sequence presented as SEQ ID NO: 3.

In a still further embodiment, the invention provides novel polypeptidesencoded by a polynucleotide sequence capable of hybridizing under highstringency conditions with the polynucleotide sequence presented as SEQID NO: 8, its complementary strand, or a sub-sequence thereof. In apreferred embodiment, the polypeptide of the invention is encoded by apolynucleotide sequence being at least 70% homologous to thepolynucleotide sequence presented as SEQ ID NO: 8. In its most preferredembodiment, the polypeptide of the invention is encoded by thepolynucleotide sequence presented as SEQ ID NO: 8.

In a still further embodiment, the invention provides novel polypeptidesencoded by a polynucleotide sequence capable of hybridizing under highstringency conditions with the polynucleotide sequence presented as SEQID NO: 15, its complementary strand, or a sub-sequence thereof. In apreferred embodiment, the polypeptide of the invention is encoded by apolynucleotide sequence being at least 70% homologous to thepolynucleotide sequence presented as SEQ ID NO: 15. In its mostpreferred embodiment, the polypeptide of the invention is encoded by thepolynucleotide sequence presented as SEQ ID NO: 15.

Biological Origin

The polypeptide of the invention may be isolated from mammalian cells,preferably from a human cell or from a cell of murine origin.

In a most preferred embodiment, the polypeptide of the invention may beisolated from human heart tissue, from human skeletal muscle, from humanpancreas, or from human brain tissue, in particular from caudate nucleusor from thalamus, or it may be obtained from DNA of mammalian origin, asdiscussed in more detail below.

Neurotrophic Activity

Neublastin polypeptides of the invention are useful for moderatingmetabolism, growth, differentiation, or survival of a nerve or neuronalcell. In particular, neublastin polypeptides are used to treating or toalleviate a disorder or disease of a living animal, e.g., a human, whichdisorder or disease is responsive to the activity of a neurotrophicagents. Such treatments and methods are described in more details below.

Antibodies

Neublastin polypeptides or polypeptide fragments of the invention areused to produce neublastin-specific antibodies. As used herein, a“neublastin-specific antibody is an antibody, e.g., a polyclonalantibody or a monoclonal antibody, that is immunoreactive to aneublastin polypeptide or polypeptide fragment, or that binds withspecificity to an epitopes of a neublastin polypeptides.

The preparation of polyclonal and monoclonal antibodies is well known inthe art. Polyclonal antibodies may in particular be obtained asdescribed by, e.g., Green et al.,: “Production of Polyclonal Antisera”in Immunochemical Protocols (Manson, Ed.); Humana Press, 1992, pages1-5; by Coligan et al.,: “Production of Polyclonal Antisera in Rabbits,Rats, Mice and Hamsters” in Current Protocols in Immunology, 1992,Section 2.4.1, and by Ed Harlow and David Lane (Eds.) in “Antibodies; Alaboratory manual” Cold Spring Harbor Lab. Press 1988. These protocolsare hereby incorporated by reference. Monoclonal antibodies may inparticular be obtained as described by, e.g., Kohler & Milstein, Nature.1975, 256:495; Coligan et al., in Current Protocols in Immunology, 1992,Sections 2.5.1-2.6.7; and Harlow et al., in Antibodies: A LaboratoryManual; Cold Spring Harbor, Pub., 1988, page 726; which protocols arehereby incorporated by reference.

Briefly, monoclonal antibodies may be obtained by injecting, e.g., micewith a composition comprising an antigen, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B lymphocytes, fusing the B lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive clonesthat produce the antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques, including affinitychromatography with protein A Sepharose, size-exclusion chromatography,and ion-exchange chromatography, see. e.g. Coligan et al. in CurrentProtocols in Immunology, 1992, Sections 2.7.1-2.7.12, and Sections2.9.1-2.9.3; and Barnes et al.: “Purification of Immunoglobulin G (IgG)”in Methods in Molecular Biology; Humana Press, 1992, Vol. 10, Pages79-104. Polyclonal or monoclonal antibodies may optionally be furtherpurified, e.g. by binding to and elution from a matrix to which thepolypeptide, to which the antibodies were raised, is bound.

Antibodies which bind to the neublastin polypeptide of the invention canbe prepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunising antigen. The polypeptide used toimmunise an animal may be obtained by recombinant DNA techniques or bychemical synthesis, and may optionally be conjugated to a carrierprotein. Commonly used carrier proteins which are chemically coupled tothe peptide include keyhole limpet hemocyanin (KLH), thyroglobulin,bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide maythen be used to immunise the animal, which may in particular be a mouse,a rat, a hamster or a rabbit.

In one embodiment, antibodies are produced using the following peptides:Peptide 1: CRPTRYEAVSFMDVNST (amino acids 108-124 of SEQ ID NO: 9); orPeptide 2: ALRPPPGSRPVSQPC (amino acids 93-107 of SEQ ID NO: 9). Methodsfor producing antibodies using these polypeptides are described inExample 10.

We also generated rabbit polyclonal antibodies to the followingpeptides:

Peptide R27: GPGSRARAAGARGC (amino acids 30-43 of SEQ ID NO:9);

Peptide R28: LGHRSDELVRFRFC (amino acids 57-70 of SEQ ID NO:9);

Peptide R29: CRRARSPHDLSL (amino acids 74-85 of SEQ ID NO:9);

Peptide R30: LRPPPGSRPVSQPC (amino acids 94-107 of SEQ ID NO:9); and

Peptide R31: STWRTVDRLSATAC (amino acids 123-136 of SEQ ID NO:9).

Of this group, only peptides R30 and R31, relatively close to theC-terminus, recognized the denatured protein under reducing conditionson a Western blot.

We have also identified additional neublastin-derived peptides derivedfrom the mature protein, as detailed below, which are predicted surfaceexposed loops based on the known GDNF structure (Eigenbrot and Gerber,Nat. Struct. Biol., 4, pp. 435-438 (1997)), and are thus useful forantibody generation:

Region 1: CRLRSQLVPVRALGLGHRSDELVRFRFC (AA43-70 of SEQ. ID. NO: 9)

Region 2: CRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC (AA74-107 of SEQ. ID. NO:9)

Region 3: CRPTRYEAVSFMDVNSTWRTVDRLSATAC (AA108-136 of SEQ. ID. NO: 9)

In another aspect of the invention, antibodies which specifically bindneublastin or neublastin-derived peptides may be used for detecting thepresence of such neublastin neurotrophic factors in various media, andin particular for the diagnosis of conditions or diseases associatedwith the neublastin molecules of the invention. A variety of protocolsfor such detection, including ELISA, RIA and FACS, are known in the art.

The antibodies of this invention may also be used for blocking theeffect of the neurotrophic factor, and may in particular be neutralizingantibodies.

Methods of Producing the Polypeptides of the Invention

A cell comprising a DNA sequence encoding a neublastin polypeptide ofthe invention is cultured under conditions permitting the production ofthe polypeptide, followed by recovery of the polypeptide from theculture medium, as detailed below. When cells are to be geneticallymodified for the purposes of producing a neublastin polypeptide, thecells may be modified by conventional methods or by gene activation.

According to conventional methods, a DNA molecule that contains aneublastin cDNA or genomic DNA sequence may be contained within anexpression construct and transfected into cells by standard methodsincluding, but not limited to, liposome-, polybrene-, or DEAEdextran-mediated transfection, electroporation, calcium phosphateprecipitation, microinjection, or velocity driven microprojectiles(“biolistics”). Alternatively, one could use a system that delivers DNAby viral vector. Viruses known to be useful for gene transfer includeadenoviruses, adeno-associated virus, lentivirus, herpes virus, mumpsvirus, poliovirus, retroviruses, Sindbis virus, and vaccinia virus suchas canary pox virus, as well as Baculovirus infection of insect cells,in particular SfP9 insect cells.

Alternatively, the cells may be modified using a gene activation (“GA”)approach, such as described in U.S. Pat. Nos. 5,733,761 and 5,750,376,each incorporated herein by reference.

Accordingly, the term “genetically modified,” as used herein inreference to cells, is meant to encompass cells that express aparticular gene product following introduction of a DNA moleculeencoding the gene product and/or regulatory elements that controlexpression of a coding sequence for the gene product. The DNA moleculemay be introduced by gene targeting, allowing incorporation of the DNAmolecule at a particular genomic site.

Recombinant Expression Vectors

In a further aspect the invention provides a recombinant expressionvector comprising the polynucleotide of the invention. The recombinantexpression vector of the invention may be any suitable eukaryoticexpression vector. Preferred recombinant expression vectors are theubiquitin promoter containing vector pTEJ-8 (FEBS Lett. 1990 267289-294), and derivatives hereof, e.g. pubi1Z. A preferred commerciallyavailable eukaryotic expression vectors is e.g. the virus promotercontaining vector pcDNA-3 (available from Invitrogen). Another preferredexpression vector uses SV40 early and adenovirus major late promoters(derived from plasmid pAD2beta; Norton and Coffin, Mol. Cell. Biol. 5:281 (1985)).

This invention also provides prokaryotic expression vectors andsynthetic genes (syngenes) with codon optimization for prokaryoticexpression. Syngenes were constructed with lower GC content andpreferred bacterial (e.g., E. coli) codons. The syngene is being clonedinto two vectors, pET19b and pMJB164, a derivative of pET19b. Theconstruction with pET19b is shown in FIG. 14. In this construct, thesequence encoding the mature domain of neublastin is directly fused toan initiating methionine. The construction with pMJB164 is shown in FIG.15.

Production Cells

In a yet further aspect the invention provides a production cellgenetically manipulated to comprise the isolated polynucleotide sequenceof the invention, and/or or a recombinant expression vector of theinvention. The cell of the invention may in particular be geneticallymanipulated to transiently or stably express, over-express or co-expresspolypeptide of the invention. Methods for generating transient andstable expression are known in the art.

The polynucleotide of the invention may be inserted into an expressionvector, e.g. a plasmid, virus or other expression vehicle, andoperatively linked to expression control sequences by ligation in a waythat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. Suitable expressioncontrol sequences include promoters, enhancers, transcriptionterminators, start codons, splicing signals for introns, and stopcodons, all maintained in the correct reading frame of thepolynucleotide of the invention so as to permit proper translation ofmRNA. Expression control sequences may also include additionalcomponents such as leader sequences and fusion partner sequences.

The promoter may in particular be a constitutive or an induciblepromoter. When cloning in bacterial systems, inducible promoters such aspL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter), maybe used. When cloning in mammalian systems, promoters derived from thegenome of mammalian cells, e.g. the ubiquitin promoter, the TK promoter,or the metallothionein promoter, or from mammalian viruses, e.g. theretrovirus long terminal repeat, the adenovirus late promoter or thevaccinia virus 7.5K promoter, may be used. Promoters obtained byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the polynucleotide of the invention.

Suitable expression vectors typically comprise an origin of expression,a promoter as well as specific genes which allow for phenotypicselection of the transformed cells, and include vectors like theT7-based expression vector for expression in bacteria [Rosenberg et al;Gene 1987 56 125], the pTEJ-8, pUbi1Z, pcDNA-3 and pMSXND expressionvectors for expression in mammalian cells [Lee and Nathans, J. Biol.Chem. 1988 263 3521], baculovirus derived vectors for expression ininsect cells, and the oocyte expression vector PTLN [Lorenz C, Pusch M &Jentsch T J: Heteromultimeric CLC chloride channels with novelproperties; Proc. Natl. Acad. Sci. USA 1996 93 13362-13366].

In a preferred embodiment, the cell of the invention is an eukaryoticcell, e.g., a mammalian cell, e.g., a human cell, an oocyte, or a yeastcell. The cell of the invention may be without limitation a humanembryonic kidney (HEK) cell, e.g., a HEK 293 cell, a BHK21 cell, aChinese hamster ovary (CHO) cell, a Xenopus laevis oocyte (XLO) cell. Inanother embodiment, the cell of the invention is a fungal cell, e.g., afilamentous fungal cell. In another preferred embodiment, the cell is aninsect cell, most preferably the Sf9 cell. Additional preferredmammalian cells of the invention are PC12, HiB5, RN33b cell lines andhuman neural progenitor cells. Most preferred are human cells.

Examples of primary or secondary cells include fibroblasts, epithelialcells including mammary and intestinal epithelial cells, endothelialcells, formed elements of the blood including lymphocytes and bonemarrow cells, glial cells, hepatocytes, keratinocytes, muscle cells,neural cells, or the precursors of these cell types. Examples ofimmortalized human cell lines useful in the present methods include, butare not limited to, Bowes Melanoma cells (ATCC Accession No. CRL 9607),Daudi cells (ATCC Accession No. CCL 213), HeLa cells and derivatives ofHeLa cells (ATCC Accession Nos. CCL 2, CCL 2.1, and CCL 2.2), HL-60cells (ATCC Accession No. CCL 240), HT-1080 cells (ATCC Accession No.CCL 121), Jurkat cells (ATCC Accession No. TIB 152), KB carcinoma cells(ATCC Accession No. CCL 17), K-562 leukemia cells (ATCC Accession No.CCL 243), MCF-7 breast cancer cells (ATCC Accession No. BTH 22), MOLT-4cells (ATCC Accession No. 1582), Namalwa cells (ATCC Accession No. CRL1432), Raji cells (ATCC Accession No. CCL 86), RPMI 8226 cells (ATCCAccession No. CCL 155), U-937 cells (ATCC Accession No. CRL 1593),WI-38VA13 sub line 2R4 cells (ATCC Accession No. CLL 75.1), and 2780ADovarian carcinoma cells (Van der Blick et al., Cancer Res. 48:5927-5932, 1988), as well as heterohybridoma cells produced by fusion ofhuman cells and cells of another species. Secondary human fibroblaststrains, such as WI-38 (ATCC Accession No. CCL 75) and MRC-5 (ATCCAccession No. CCL 171), may also be used.

When the cell of the invention is an eukaryotic cell, incorporation ofthe heterologous polynucleotide of the invention may be in particular becarried out by infection (employing a virus vector), by transfection(employing a plasmid vector), using calcium phosphate precipitation,microinjection, electroporation, lipofection, or other physical-chemicalmethods known in the art.

In a more preferred embodiment the isolated polynucleotide sequence ofthe invention, and/or or a recombinant expression vector of theinvention are transfected in a mammalian host cell, a neural progenitorcell, an astrocyte cell, a T-cell, a hematopoitic stem cell, anon-dividing cell, or a cerebral endothelial cell, comprising at leastone DNA molecule capable of mediating cellular immortalization and/ortransformation.

Activation of an endogenous gene in a host cell may be accomplished bythe introducing regulatory elements, in particular by the introducing apromoter capable of effecting transcription of an endogenous geneencoding the neublastin polypeptide of the invention.

Pharmaceutical Compositions

In another aspect the invention provides novel pharmaceuticalcompositions comprising a therapeutically effective amount of thepolypeptide of the invention.

For use in therapy the polypeptide of the invention may be administeredin any convenient form. In a preferred embodiment, the polypeptide ofthe invention is incorporated into a pharmaceutical composition togetherwith one or more adjuvants, excipients, carriers and/or diluents, andthe pharmaceutical composition prepared by the skilled person usingconventional methods known in the art.

Such pharmaceutical compositions may comprise the polypeptide of theinvention, or antibodies hereof. The composition may be administeredalone or in combination with at one or more other agents, drugs orhormones.

The pharmaceutical composition of this invention may be administered byany suitable route, including, but not limited to oral, intravenous,intramuscular, inter-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcultaneous, intraperitoneal,intranasal, anteral, topical, sublingual or rectal application, buccal,vaginal, intraorbital, intracerebral, intracranial, intraspinal,intraventricular, intracisternal, intracapsular, intrapulmonary,transmucosal, or via inhalation.

Intrapulmonary delivery methods, apparatus and drug preparation aredescribed, for example, in U.S. Pat. Nos. 5,785,049, 5,780,019, and5,775,320, each incorporated herein by reference. Administration may beby periodic injections of a bolus of the preparation, or may be mademore continuous by intravenous or intraperitoneal administration from areservoir which is external (e.g., an IV bag) or internal (e.g., abioerodable implant, a bioartificial organ, or a colony of implantedneublastin production cells). See, e.g., U.S. Pat. Nos. 4,407,957,5,798,113, and 5,800,828, each incorporated herein by reference.Intrapulmonary delivery methods and apparatus are described, forexample, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference.

In particular, administration of a neublastin according to thisinvention may be achieved using any suitable delivery means, including:

(a) pump (see, e.g., Annals of Pharmacotherapy, 27:912 (1993); Cancer,41:1270 (1993); Cancer Research, 44:1698 (1984), incorporated herein byreference),

(b), microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888;and 5,084,350, herein incorporated by reference),

(c) continuous release polymer implants (see, e.g., Sabel, U.S. Pat. No.4,883,666, incorporated herein by reference),

(d) macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881,4,976,859 and 4,968,733 and published PCT patent applicationsWO92/19195, WO 95/05452, each incorporated herein by reference);

(e) naked or unencapsulated cell grafts to the CNS (see, e.g., U.S. Pat.Nos. 5,082,670 and 5,618,531, each incorporated herein by reference); or

(f) injection, either subcutaneously, intravenously, intra-arterially,intramuscularly, or to other suitable site;

(g) oral administration, in capsule, liquid, tablet, pill, or prolongedrelease formulation.

In one embodiment of this invention, a neublastin is delivered directlyinto the CNS, preferably to the brain ventricles, brain parenchyma, theintrathecal space or other suitable CNS location, most preferablyintrathecally.

In another preferred embodiment, we contemplate systemic delivery bysubcutaneous injection, intravenous administration, or intravenousinfusion.

Other useful parenteral delivery systems include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, pumpdelivery, encapsulated cell delivery, liposomal delivery,needle-delivered injection, needle-less injection, nebulizer,aeorosolizer, electroporation, and transdermal patch.

Further details on techniques for formulation and administration may befound in the latest edition of Reminzton's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

The active ingredient may be administered in one or several doses perday. Currently contemplated appropriate dosages are between 0.5 ngneublastin/kg body weight to about 50 μg/kg per administration, and fromabout 1.0 ng/kg to about 100 μg/kg daily. The neublastin pharmaceuticalcomposition should provide a local concentration of neurotrophic factorof from about 5 ng/ml cerebrospinal fluid (“CSF”) to 25 ng/ml CSF.

The dose administered must of course be carefully adjusted to the age,weight and condition of the individual being treated, as well as theroute of administration, dosage form and regimen, and the resultdesired, and the exact dosage should of course be determined by thepractitioner.

In further embodiments, the Neublastin polypeptide of the invention maybe administered by genetic delivery, using cell lines and vectors asdescribed below under methods of treatment. To generate such therapeuticcell lines, the polynucleotide of the invention may be inserted into anexpression vector, e.g. a plasmid, virus or other expression vehicle,and operatively linked to expression control sequences by ligation in away that expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. Suitable expressioncontrol sequences include promoters, enhancers, transcriptionterminators, start codons, splicing signals for introns, and stopcodons, all maintained in the correct reading frame of thepolynucleotide of the invention so as to permit proper translation ofmRNA. Expression control sequences may also include additionalcomponents such as leader sequences and fusion partner sequences.

The promoter may in particular be a constitutive or an induciblepromoter. Constitutive promoters could be synthetic, viral or derivedfrom the genome of mammalian cells, e.g. the human ubiquitin promoter.In a preferred embodiment the therapeutic cell line will be a humanimmortalised neural cell line expressing the polypeptide of theinvention. For implantation, we contemplate implanting between about 10⁵to 10¹⁰ cells, more preferably 10⁶ to about 10⁸ cells.

Methods of Treatment

The present invention, which relates to polynucleotides and proteins,polypeptides, peptide fragments or derivatives produced therefrom, aswell as to antibodies directed against such proteins, peptides orderivatives, may be used for treating or alleviating a disorder ordisease of a living animal body, including a human, which disorder ordisease is responsive to the activity of neurotrophic agents.

The polypeptides of the present invention may be used directly via,e.g., injected, implanted or ingested pharmaceutical compositions totreat a pathological process responsive to the neublastin polypeptides.

The polynucleotide of the invention, including the complementarysequences thereof, may be used for the expression of the neurotrophicfactor of the invention. This may be achieved by cell lines expressingsuch proteins, peptides or derivatives of the invention, or by virusvectors encoding such proteins, peptides or derivatives of theinvention, or by host cells expressing such proteins, peptides orderivatives. These cells, vectors and compositions may be administeredto treatment target areas to affect a disease process responsive to theneublastin polypeptides.

Suitable expression vectors may be derived from lentiviruses,retroviruses, adenoviruses, herpes or vaccinia viruses, or from variousbacterially produced plasmids may be used for in vivo delivery ofnucleotide sequences to a whole organism or a target organ, tissue orcell population. Other methods include, but are not limited to, liposometransfection, electroporation, transfection with carrier peptidescontaining nuclear or other localizing signals, and gene delivery viaslow-release systems. In still another aspect of the invention,“antisense” nucleotide sequences complementary to the neublastin gene orportions thereof, may be used to inhibit or enhance neublastinexpression.

In yet another aspect the invention relates to a method of treating oralleviating a disorder or disease of a living animal body, including ahuman, which disorder or disease is responsive to the activity ofneurotrophic agents.

The disorder or disease may in particular be damages of the nervoussystem caused by trauma, surgery, ischemia, infection, metabolicdiseases, nutritional deficiency, malignancy or toxic agents, andgenetic or idiopathic processes.

The damage may in particular have occurred to sensory neurons or retinalganglion cells, including neurons in the dorsal root ganglion or in anyof the following tissues: The geniculate, petrosal and nodose ganglia;the vestibuloacoustic complex of the VIIIth cranial nerve; theventrolateral pole of the maxillomandribular lobe of the trigeminalganglion; and the mesencephalic trigeminal nucleus.

In a preferred embodiment of the method of the invention, the disease ordisorder is a neurodegenerative disease involving lesioned and traumaticneurons, such as traumatic lesions of peripheral nerves, the medulla,and/or the spinal cord, cerebral ischaemic neuronal damage, neuropathyand especially peripheral neuropathy, peripheral nerve trauma or injury,ischemic stroke, acute brain injury, acute spinal cord injury, nervoussystem tumors, multiple sclerosis, exposure to neurotoxins, metabolicdiseases such as diabetes or renal dysfunctions and damage caused byinfectious agents, neurodegenerative disorders including Alzheimer'sdisease, Huntington's disease, Parkinson's disease, Parkinson-Plussyndromes, progressive Supranuclear Palsy (Steele-Richardson-OlszewskiSyndrome), Olivopontocerebellar Atrophy (OPCA), Shy-Drager Syndrome(multiple systems atrophy), Guamanian parkinsonism dementia complex,amyotrophic lateral sclerosis, or any other congenital orneurodegenerative disease, and memory impairment connected to dementia.

In a preferred embodiment, we contemplate treatment of sensory and/orautonomic system neurons. In another preferred embodiment, wecontemplate treatment of motor neuron diseases such as amyotrophiclateral sclerosis (“ALS”) and spinal muscular atrophy. In yet anotherpreferred embodiment, we contemplate use of the neublastin molecules ofthis invention to enhance nerve recovery following traumatic injury. Inone embodiment we contemplate use of a nerve guidance channel with amatrix containing neublastin polypeptides. Such nerve guidance channeslare disclosed, e.g., U.S. Pat. No. 5,834,029, incorporated herein byreference.

In a preferred embodiment, the polypeptides and nucleic acids of thisinvention (and pharmaceutical compositions containing same) are used inthe treatment of peripheral neuropathies. Among the peripheralneuropathies contemplated for treatment with the molecules of thisinvention are trauma-induced neuropathies, e.g., those caused byphysical injury or disease state, physical damage to the brain, physicaldamage to the spinal cord, stroke associated with brain damage, andneurological disorders related to neurodegeneration.

We also contemplate treatment of chemotherapy-induced neuropathies (suchas those caused by delivery of chemotherapeutic agents, e.g., taxol orcisplatin); toxin-induced neuropathies, drug-induced neuropathies,vitamin-deficiency-induced neuropathies; idiopathic neuropathies; anddiabetic neuropathies. See, e.g., U.S. Pat. Nos. 5,496,804 and5,916,555, each herein incorporated by reference.

We also contemplate treatment of mon-neuropathies, mono-multiplexneuropathies, and poly-neuropathies, including axonal and demyelinatingneuropathies, using the neublastin nucleotides and polypeptides of thisinvention.

In another preferred embodiment, the polypeptides and nucleic acids ofthis invention (and pharmaceutical compositions containing same) areused in the treatment of various disorders in the eye, includingphotoreceptor loss in the retina in patients afflicted with maculardegeneration, retinitis pigmentosa, glaucoma, and similar diseases.

Another object of the present invention is to provide a method for theprevention of the degenerative changes connected with the above diseasesand disorders, by implanting into mammalian brain including humanvectors or cells capable of producing a biologically active form ofneublastin or a precursor of neublastin, i.e. a molecule that canreadily be converted to a biologically active form of neublastin by thebody, or additionally cells that secrete neublastin may be encapsulated,e.g. into semipermeable membranes.

Cells can be grown in vitro for use in transplantation or engraftmentinto mammalian brain including human.

In a preferred embodiment, the gene encoding the polypeptide of theinvention is transfected into a suitable cell line, e.g. into animmortalised rat neural stem cell line like HiB5 and RN33b, or into ahuman immortalised neural progenitor cell line, and the resulting cellline is implanted in the brain of a living body, including a human, tosecrete the therapeutic polypeptide of the invention in the CNS, e.g.using the expression vectors described in International PatentApplication WO 98/32869.

Methods of Diagnosis and Screening

A neublastin nucleic acid can be used to determine whether an individualis predisposed to developing a neurological disorder resulting from adefect in the neublastin gene, e.g., an defect in a neublastin allele,which has been acquired by, e.g., genetic inheritance, by abnormalembryonic development, or by acquired DNA damage. The analysis can beby, e.g., detecting a deletion(s) or a point-mutation(s) within theneublastin gene, or by detecting the inheritance of such predispositionof such genetic defects with specific restriction fragment lengthpolymorphisms (RFLPs), by detecting the presence or absence of a normalneublastin gene by hybridizing a nucleic acid sample from the patientwith a nucleic acid probe(s) specific for the neublastin gene, anddetermining the ability of the probe to hybridize to the nucleic acid.

In particular, a neublastin nucleic acid can be used as a hybridizationprobe. Such hybridization assays may be used to detect, prognose,diagnose, or monitor the various conditions, disorders, or diseasestates associated with aberrant levels of the mRNAs encoding theNeublastin protein. A neublastin nucleic acid can be construed as a“marker” for neublastin neurotrophic factor-dependant physiologicalprocesses. These processes include, but are not limited to, “normal”physiological processes (e.g., neuronal function) and pathologicalprocesses (e.g., neurodegenerative disease). The characterization of aparticular patient sub-population(s) with aberrant (i.e., elevated ordeficient) levels of the neublastin protein and or neublastin-encodingmRNA may lead to new disease classifications. By “aberrant levels,” asdefined herein, is meant an increased or decreased level relative tothat in a control sample or individual not having the disorderdetermined by quantitative or qualitative means.

The neublastin nucleic acids and polypeptides of this invention may alsobe used to screen for and identify neublastin analogs, including smallmolecule mimetics of neublastin. In one contemplated embodiment, theinvention provides a method for identifying a candidate compound thatinduces a neuroblastin-mediated biological effect, the method comprisingthe steps of providing a test cell which when contacted with neublastinis induced to express a detectable product, exposing the cell to thecandidate compound, and detecting the detectable product. The expressionof the detectable product is indicative of the ability of the candidatecompound to induce the neuroblastin-mediated biological effect.

Further, the neublastin nucleic acids and polypeptides of this inventionmay be used on DNA chip or protein chips,or in computer programs toidentify related novel gene sequences and proteins encoded by them,including allelic variants and single nucleotide polymorphisms (“SNPs”).Such methods are described, e.g., in U.S. Pat. Nos. 5,795,716;5,754,524; 5,733,729; 5,800,992; 5,445,934; 5,525,464, each hereinincorporated by reference.

EXAMPLES Example 1

Methods for Isolating Neublastin Nucleic Acids

Method 1

Rapid-Screening of Human Fetal Brain cDNA for the Neublastin Gene

A 290 bp fragment was identified in two high throughput genomicsequences (HGTS) submitted to GenBank (Accession No. AC005038 andAC005051) by its homology to human persephin. From the nucleic acidsequence of the 290 bp fragment, two neublastin specific primers weresynthesized. The neublastin top strand primer (“NBNint.sence”) had thesequence 5′-CCT GGC CAG CCT ACT GGG-3′ (SEQ. ID. NO.: 17). Theneublastin bottom strand primer (“NBNint.antisence”) had the sequence5′-AAG GAG ACC GCT TCG TAG CG-3′ (SEQ. ID.

NO.: 18). With these primers, 96-well PCR reactions were performed.

A 96-well master plate, containing plasmid DNA from 500,000 cDNA clones,was loaded with approximately 5000 clones per well. A 96-well sub-platewas utilized with E.coli DH10B glycerol stock containing 50 clones perwell.

A neublastin nucleic acid was identified by three rounds ofamplification using polymerase chain reaction (“PCR”) techniques;amplification increases the number of copies of the nucleic acid in thesample.

Master Plate Screening: Using the 96-well PCR screening techniquedescribed above, a human fetal brain cDNA master plate was screened withthe gene-specific primers to isolate the human neublastin cDNA.

Thirty nanograms (30 ng) of human fetal brain cDNA (6 ng/μl; OrigeneTechnologies) was obtained from the corresponding well of the masterplate and placed in a total volume of 25 μl which contained thefollowing reagents: 0.2 mM of each of the two aforementionedgene-specific primers (i.e., NBNint.sence and NBNint.antisence),1×standard PCR buffer (Buffer V, Advanced Biotechnologies, UK), 0.2 mMdNTPs (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA);and 0.5 units of Taq DNA polymerase (5 U/μl; Advanced Biotechnologies,UK).

PCR thermocycling reactions were performed using the following procedureand conditions. DNA was initially denatured at 94° C. for 3 minutes, andthen followed by 35 cycles of denaturation at 94° C. for 1 minute each,annealing at 55° C. for 1 minute, a first extension at 72° C. for 90seconds; and a final extension at 72° C. for 5 minutes. The products of96 individual PCR reactions were analysed by gel electrophoresis using a2% agarose gel containing ethidium bromide stain. The 102 bp, positivePCR product seen from a well was found to correspond to a unique 96-wellsub-plate.

The 102 bp nucleic acid fragment had the following sequence [SEQ ID NO.13]:

5′-CCTGGCCAGCCTACTGGGCGCCGGGGCCCTGCGACCGCCCCCGGGCTCCCGGCCCGTCAGCCAGCCCTGCTGCCGACCCACGCGCTACGAAGCGGTCTCCTT-3′

Sub-Plate Screening: The 96-well human fetal brain sub-plate wasscreened by PCR-mediated amplification by placing 1 μl of the glycerolstock from the corresponding sub-plate well in a total volume of 25 μlwhich contained: 0.2 mM of each of the two gene-specific primers;1×standard PCR buffer (Buffer V; Advanced Biotechnologies, UK); 0.2 mMdNTPs (Amersham-Pharmacia); 0.1 M GC-Melt (Clontech Laboratories, USA);and 0.5 units of Taq DNA polymerase (5 U/μl; Advanced Biotechnologies,UK).

The same PCR thermocycling conditions as described for the masterplatescreening were utilized. The 96 individual PCR reactions were analysedon a 2% agarose gel containing ethidium bromide and a positive well wasidentified which gave the 102 bp PCR fragment.

Colony PCR: One ml of the glycerol stock from the positive sub-platewell was diluted 1:100 in Luria broth (LB). One ml and 10 ml of theaforementioned dilution were then plated on two separate agar platescontaining Luria broth (“LB”), and 100 μg/ml carbenicillin. The LBplates were then incubated overnight at 30° C. From these plates, 96colonies were picked into a new 96-well PCR plate containing: 0.2 mM ofeach of the two aforementioned gene-specific primers, 1× standard PCRbuffer (Buffer V; Advanced Biotechnologies, UK), 0.2 mM dNTPs(Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA), and0.5 units of Taq DNA polymerase (5 U/μl; Advanced Biotechnologies, UK)in a final volume of 25μl.

The same PCR thermocycling conditions as described for the masterplatescreening were utilized. The 96 individual PCR reactions were thenanalysed on a 2% agarose gel containing ethidium bromide. A positivecolony containing the 102 bp fragment was subsequently identified.

Sequencing of the plasmid DNA prepared from this positive colonyrevealed a full-length cDNA of 861 bp [SEQ ID NO: 8]. The cDNA coded fora pre-pro-neublastin [SEQ ID NO: 9]. Automated DNA sequencing wasperformed using the BigDye® terminator cycle sequencing kit (PE AppliedBiosystems, USA). The sequencing gels were run on the ABI Prism 377 (PEApplied Biosystems, USA).

Method 2

Cloning Neublastin cDNA from Human Brain

An additional method of amplifying the full-length neublastin cDNA orcDNA fragment can be performed by RACE (Rapid Amplification of cDNAends) and the neublastin-specific primers NBNint.sence andNBNint.antisence described above, combined with vector-specific oradapter-specific primers, for example by using the Marathon cDNAamplification kit (Clontech Laboratories, USA, Cat. No. K1802-1).

Whole human brain Marathon-Ready cDNA (Clontech Laboratories, USA,Catalogue. No. 7400-1) can be used to amplify the full-length neublastincDNA. Useful primers for amplification include a neublastin top strandprimer 5′-ATGGAACTTGGACTTGG-3′ (SEQ ID NO.: 19) (“NBNext.sence”), and aneublastin bottom strand primer 5′-TCCATCACCCACCGGC-3′ (SEQ ID NO.: 20)(“NBNext.antisence”), combined with the adaptor primer AP1 included withthe Marathon-Ready cDNA. An alternative top strand primer has also beenused, 5′-CTAGGAGCCCATGCCC-3′ (SEQ ID NO.: 28). A further alternativebottom strand primer, 5′-GAGCGAGCCCTCAGCC-3′ (SEQ ID NO.: 33) may alsobe used. Likewise, alternative bottom strand primers SEQ ID NOS.: 24 and26 may also be used.

Method 3

Cloning Neublastin cDNA from Human Brain

Another method of cloning neublastin cDNA is by screening human adult orfetal brain libraries with one or more neublastin probes describedherein (and as exemplified in FIG. 1). or These libraries include: λgt11human brain (Clontech Laboratories, USA, Cat. No. HL3002b); or λgt11human fetal brain (Clontech Laboratories, USA, Cat. No. HL3002b).

Method 4

Rapid-Screening of Mouse Fetal cDNA for the Neublastin Gene

A rapid screening procedure for the neublastin gene was performed in thefollowing manner. A 96-well master plate, containing plasmid DNA from500,000 cDNA clones, was loaded with approximately 5000 clones per well.A 96-well sub-plate was utilized with E. Coli glycerol stock containing50 clones per well. Three rounds of PCR-mediated amplification wasperformed in order to identify a gene of interest (i.e., neublastin).

Master Plate Screening: A mouse fetal cDNA master plate was screened by96-well PCR using gene-specific primers to isolate the mouse neublastincDNA. The following two primers were synthesised:

(1) neublastin C2 primer (NBNint.sence): 5′-GGCCACCGCTCCGACGAG-3′ (SEQID NO: 21); and (2) neublastin C2as primer (NBNint.antisence):

5′-GGCGGTCCACGGTTCTCCAG-3′ (SEQ ID NO: 22).

By using these two gene-specific primers a 220 bp positive PCR productwas identified. The 220 bp nucleic acid possessed the following sequence[SEQ ID NO. 14]:

 5′-GGCCACCGCTCCGACGAGCTGATACGTTTCCGCTTCTGCAGCGGCTCGTGCCGCCGAGCACGCTCCCAGCACGATCTCAGTCTGGCCAGCCTACTGGGCGCTGGGGCCCTACGGTCGCCTCCCGGGTCCCGGCCGATCAGCCAGCCCTGCTGCCGGCCCACTCGCTATGAGGCCGTCTCCTTCATGGACGTGAACAGCACCTGGAGAACCGTGGACCGCC-3′

96-well PCR reactions were then performed in the following manner.Thirty nanograms of mouse fetal brain cDNA (6 ng/μl; OrigeneTechnologies) was obtained from the corresponding well of the masterplate and placed in a total volume of 25 μl which also contained: 0.2 mMof each of the two aforementioned gene-specific primers (i.e., C2 primer(NBNint.sence) and neublastin C2as primer (NBNint.antisence)),1×standard PCR buffer (Buffer V; Advanced Biotechnologies, UK), 0.2 mMdNTPs (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA),and 0.5 units of Taq DNA polymerase (5 U/μl; Advanced Biotechnologies,UK).

The following PCR thermocycling conditions were utilized: an initialdenaturation at 94° C. for 3 minutes, followed by 35 cycles ofdenaturation at 94° C. for 1 minute each, annealing at 55° C. for 1minute, extension at 72° C. for 90 seconds; and a final extension at 72°C. for 5 minutes. The 96 individual PCR reactions were analysed on a 2%agarose gel containing ethidium bromide stain. The 220 bp, positive PCRproduct seen from a well was found to correspond to a unique 96-wellsub-plate. The 96 individual PCR reactions were then analysed by gelelectrophoresis on a 2% agarose gel containing ethidium bromide stain.The 220 bp positive PCR product which had been identified correspondedto a unique well of the 96-well sub-plate.

Sub-Plate Screening: The 96-well mouse fetal sub-plate was screened byPCR-mediated amplification by placing 1 μl of the glycerol stock fromthe corresponding sub-plate well into a final, total volume of 25 μlwhich contained: 0.2 mM of each of the two aforementioned gene-specificprimers; 1×standard PCR buffer (Buffer V; Advanced Biotechnologies, UK);0.2 mM dNTPs (Amersham-Pharmacia); 0.1 M GC-Melt (Clontech Laboratories,USA); and 0.5 units of Taq DNA polymerase (5 U/μl; AdvancedBiotechnologies, UK). The PCR thermocycling was performed according tothe conditions described above for the master plate screening.

The individual 96 PCR reactions were then analysed on a 2% agarose gelcontaining ethidium bromide and a positive well was identified whichproduced the 220 bp fragment.

Colony PCR: One ml of the glycerol stock from the positive sub-platewell was diluted 1:100 in Luria broth (LB). One ml and 10 ml of theaforementioned dilution were then plated on two separate LB plates,containing 100 μg/ml carbenicillin, and incubated at 30° C. overnight. Atotal of 96 colonies were isolated and transferred to a 96-well PCRplate containing: 0.2 mM of each of the two aforementioned gene-specificprimers, 1×standard PCR buffer (Buffer V; Advanced Biotechnologies, UK),0.2 mM dNTPs (Amersham-Pharmacia); 0.1 M GC-Melt (Clontech Laboratories,USA), and 0.5 units of Taq DNA polymerase (5 U/μl; AdvancedBiotechnologies UK) in a final volume of 25 μl.

PCR thermocycling was performed according to the conditions describedabove (see, “master plate screening”, infra). The 96 individual PCRreactions were analysed by gel electrophoresis on a 2% agarose gelcontaining ethidium bromide. A positive colony was identified by thepresence of the 220 bp fragment. Plasmid DNA was prepared from thispositive colony. The clone was sequenced by automated DNA sequencingusing the BigDye® terminator cycle sequencing kit with AmpliTaq DNApolymerase. The sequencing gels were run on the ABI Prism 377 (PEApplied Biosystems). The resulting sequence of this clone revealed afull-length cDNA of 2136 bp (SEQ ID NO: 15). The cDNA includes an openreading frame with the predicted amino acid sequence shown in SEQ ID NO:16, which codes for a mouse pre-pro-neublastin polypeptide.

Example 2

Cloning of Genomic Neublastin

As discussed above, applicants identified a 290 bp nucleic acid fragmentin two human BAC clones with entries in GenBank (with the Accession Nos.AC005038 and AC005051) which had regions of homology to persephin and tothe flanking sequences of persephin. Applicants used the 861 bppredicted sequence described above to design additional primers, withthe goal of cloning a nucleic acid encoding additional neublastinnucleic acids using Lasergene Software (DNAStar, Inc.). Two pairs ofprimers were used to clone the neublastin gene by using PCR reactions ongenomic DNA. The two pairs of primers are illustrated below.

Primer Pair No. 1

5′ CCA AgC CCA CCT ggg TgC CCT CTT TCT CC 3′ (sense)  (SEQ ID NO:23).

5′ CAT CAC CCA CCg gCA ggg gCC TCT CAg 3′ (antisense)  (SEQ ID NO:24).

Primer Pair No. 2

5′ gAgCCCAtgCCCggCCTgATCTCAgCCCgA ggACA 3′ (sense)  (SEQ ID NO:25).

5′ CCCTggCTgAggCCgCTggCTAgTgggACTCTgC 3′ (antisense)  (SEQ ID NO:26).

Using primer pair No. 1, a 887 bp DNA fragment was amplified from apreparation of human genomic DNA purchased from Clontech Laboratories,(Cat. No. 6550-1).

PCR protocol: PCR was performed using the Expand High Fidelity PCRsystem (Boehringer Mannheim) with buffer 1. The PCR reaction mixture wassupplemented with 5% dimethylsulfoxide (DMSO) and 17.5 pmol of each dNTPin a total volume of 50 μl. Thermocycling was performed with apre-denaturation step at 94° C. for 2 minutes, followed by 35 two-stepcycles at 94° C. for 10 seconds, and 68° C. for 1 minute, respectively.Thermocycling was terminated by incubation at 68° C. for 5 minutes.Thermocycling was carried out in a PTC-225 DNA Engine Tetradthermocycler (MJ Research, MA). The PCR products were analysed by gelelectrophoresis on 2% agarose (FMC) and then photographed.

The 887 bp fragment amplified from human genomic DNA with primer pairNo. 1 was cloned into the pCRII vector (Invitrogen), and transformedinto XL 1-Blue competent E.coli cells (Stratagene). The resultingplasmid, designated neublastin-2, was sequenced using Thermosequenase(Amersham Pharmacia Biotech). Sequencing products were analysed byelectrophoreses on an ALFExpress automated sequencer (Amersham PharmaciaBiotech).

Fragments obtained by PCR amplification of human genomic DNA with thesecond pair of primers (Primer Pair No. 1, above), were sequenced,revealing an additional 42 bp region at the 3′ prime end of the openreading frame. The full-length sequence was analysed by comparing it tothe sequences of nucleic acids of other neurotrophic factors, as well asby mapping exon-intron boundaries using gene-finding software programswhich identify probable splice junctions and regions of high codingpotential using Netgene and Gene Mark software (Brunak et al., J.Mol.Biol., 220, pp. 49-65 (1991); Borodovsky et al., Nucl. Acids Res., 23,pp. 3554-62 (1995)). The exon-intron boundaries were confirmed by thecDNA obtained from the Rapid Screen described above.

As illustrated in FIG. 7, the resulting neublastin gene has two exonsseparated by a 70 bp intron. Together, the exons have a predicted aminoacid sequence of a full-length Neublastin polypeptide. The predictedcDNA (SEQ ID NO: 3) contains an open reading frame (ORF) encoding 238amino acid residues (SEQ ID NO: 4). The Neublastin-2 clone contained thecomplete coding sequence of pro-neublastin. The amino acid sequenceencoded by the gene showed high homology to three proteins, persephin,neurturin, and GDNF.

Example 3

Expression of Neublastin Nucleic Acids

Expression of neublastin RNA was detected in both nervous andnon-nervous tissue in rodents and in humans, and at variousdevelopmental immature and adult stages, using the techniques describedbelow.

Method of Detecting Neublastin RNA Expression Using RT-PCR

Based on the Neublastin DNA sequence identified as SEQ ID NO: 1, thefollowing primers were synthesised: (1) a neublastin C2 primer5′-GGCCACCGCTCCGACGAG-3′ (SEQ ID NO. 21), and (2) a neublastin C2asprimer 5′-GGCGGTCCACGGTTCTCCAG-3′ (SEQ ID NO. 22). This primer set wasused to RT-PCR amplify a DNA fragment from adult and fetal humanwhole-brain mRNA. Among the DNA fragments produced by this reaction wasone of 220 bp. Identification of this 220 bp DNA fragment confirmed thatthe neublastin gene is expressed in adult and fetal brain tissue. A 220bp DNA fragment was also amplified from genomic DNA with using theseprimers.

Method of Detecting Neublastin RNA Expression by Northern BlotHybridization

Northern blots with polyA⁺ RNA from adult human tissue were purchasedfrom a commercial supplier (Clontech Laboratories, USA) and probed witha ³²P-labeled neublastin cDNA. The labelled neublastin cDNA was preparedaccording to the methods described in Example 1, above.

Preparation of Probes: A neublastin nucleic acid DNA fragment(nucleotides 296-819 of SEQ ID NO: 8) was labelled by the Rediprime IIlabelling kit (Amersham; Cat. No. RPN1633) for use as a hybridizationprobe, as recommended by the manufacturer. Briefly, the DNA sample wasdiluted to a concentration of 2.5-25 ng in 45 μl of 10 mM TE Buffer (10mM Tris-HCl, pH 8.0, 1 mM EDTA). The DNA was then denatured by heatingthe sample to 95-100° C. for 5 minutes in a boiling water bath, quickcooling the sample by placing it on ice for 5 minutes, and then brieflycentrifuging it to bring the contents to the bottom of the reactiontube. The total amount of denatured DNA was added together with 5 μl ofRedivue [³²P] dCTP (Amersham Pharmacia Biotech Ltd.) in the reactiontube containing buffered solution of dATP, dGTP, dTTP, exonuclease freeKlenow enzyme and random primer in dried stabilised form. The solutionwas mixed by pipetting up and down 2 times, moving the pipette tiparound in the solution, and the reaction mixture was incubated at 37° C.for 10 minutes. The labelling reaction was stopped by adding 5 μl of 0.2M EDTA. For use as a hybridization probe the labelled DNA was denaturedto single strands by heating the DNA sample to 95-100° C. for 5 minutes,then snap cooling the DNA sample on ice for 5 minutes. The tube wascentrifuged and its contents mixed well. Finally the single-stranded DNAprobe was purified using the Nucleotide Removal Kit (Qiagen).

Hybridization Techniques: Prepared northern blots were purchased from acommercial supplier (“Multiple Tissue Northern Blots, ClontechLaboratories, USA, Catalogue Nos. 7760-1 and 7769-1) and were hybridizedaccording to the manufacturer's instructions using the neublastin³²P-labeled probe prepared above. For hybridization, ExpressHyb Solution(Clontech Laboratories, USA) was used, and a concentration ofapproximately 3 ng/ml of the labelled probe was employed. The ExpressHybsolution was heated to 68° C. and then stirred to dissolve anyprecipitate. Each northern blot membrane (10×10 cm) was pre-hybridizedin at least 5 ml of ExpressHyb Solution at 68° C. for 30 minutes in aHybaid Hybridization Oven according to the manufacturer's instructions.The neublastin ³²P-labeled probe was denatured at 95-100° C. for 2minutes and then chilled quickly on ice. Fourteen microliters (14 μl) ofthe labelled probe was added to 5 ml of fresh ExpressHyb, and thoroughlymixed. The ExpressHyb Solution used in the pre-hybridization wasreplaced by evenly distributing over the blots the 5 ml of freshExpressHyb Solution containing labelled DNA probe. Blots were incubatedat 68° C. for 1 hour in a Hybaid hybridization Oven. After incubation,the blots were rinsed and washed several times at low stringency (2×SSCbuffer containing 0.05% SDS at room temperature) followed by a highstringency wash (0.1×SSC containing 0.1% SDS at 50° C.) [20×SSC is 0.3 MNaCl/0.3 M Na citrate, pH 7.0]. The blots were exposed to a Hyperfilm MP(Amersham Pharmacia Biotech Ltd.) at −80° C. using intensifying screens.

The results of the northern blot hybridization experiments are presentedin FIG. 1. FIG. 1A (left) and FIG. 1B (right) are northern blots ofpolyA⁺ RNA which were probed with ³²P-labelled neublastin cDNA asdescribed in Example 3. The markers represent polynucleotides of 1.35kilobase pairs (“kb”), 2.4 kb, 4.4 kb, 7.5 kb, and 9.5 kb in size. Themembrane of FIG. 1A was prepared with mRNA extracted from various adulthuman tissues: From the results of the northern blot hybridizationanalysis, applicants conclude that neublastin mRNA is expressed in manyadult human tissues. The highest level of neublastin expression isdetected in the heart, in skeletal muscle and in the pancreas. Themembrane of FIG. 1B was prepared with RNA extracted from various regionsof the adult human brain. Within the adult brain, the highest level ofexpression is seen in the caudate nucleus and in the thalamus. An mRNAtranscript of approximately 5 kb was the predominant form of neublastinmRNA expressed in the brain.

Method of Detecting Neublastin RNA Expression Using by in situHybridization in Tissues

The following techniques are used to measure the expression ofneublastin RNA in animal tissues, e.g., rodent tissues, with aneublastin anti-sense probe.

Expression in Mice

Preparation of Tissue Samples: Time pregnant mice (B&K Universal,Stockholm, Sweden) were killed by cervical dislocation on gestationalday 13.5 or 18.5. Embryos were removed by dissection under sterileconditions, and immediately immersed in a solution of 0.1M phosphatebuffer (PB) containing 4% paraformaldehyde (“PFA”) for 24-30 hours, andthen removed from the PFA and stored in PBS. The tissue was prepared forsectioning by immersing the tissue in a solution of 30% sucrose, andthen embedding it in TissueTech (O.C.T. Compound, Sakura Finetek USA,Torrance, Calif.). Six series of coronal or sagittal sections (12 μmeach) were cut on a cryostat and thaw mounted onto positively chargedglass slides. Neonatal heads/brains (P1, P7) were fixed following thesame protocol as for the embryonic stages, and adult brain tissue wasdissected, immediately frozen on dry ice, and cut on a cryostat withoutany prior embedding.

Preparation of Neublastin Riboprobes: An antisense neublastin RNA probe(hereafter a “neublastin riboprobe”) was made as follows. Nucleotides1109-1863 of the mouse neublastin cDNA sequence (SEQ ID NO: 15) weresub-cloned into the BlueScript vector (Stratagene). The resultingplasmid was cut into a linear DNA using EcoRI restriction endonuclease.The EcoRI DNA fragment was in vitro transcribed with T3 RNA polymeraseand the digoxigenin (“DIG”) RNA Labelling Kit according to themanufacturer's instructions (Boehringer Mannheim).

Hybridization: Cryostat sections were fixed for 10 minutes in 4% PFA,treated for 5 minutes with 10 mg/ml of proteinase K, dehydratedsequentially in 70% and 95% ethanol for 5 and 2 min, respectively, andthen allowed to air dry. Hybridization buffer (50% deionized formamide,10% of a 50% dextran sulphate solution, 1% Denhardt's solution, 250μg/mlyeast tRNA, 0.3M NaCl, 20 mM Tris-HCl (pH8), 5 mM EDTA, 10 mM NAPO₄, 1%sarcosyl) containing 1 μg/ml of the DIG-labelled probe was heated to 80°C. for 2 minutes and applied onto the sections. The sections was thencovered with parafilm and incubated at 55° C. for 16-18 hours.

The next day the sections were washed at high stringency (2×SSCcontaining 50% formamide) at 55° C. for 30 minutes, and then washed inRNase buffer and incubated with 20 μg/ml of RNaseA for 30 minutes at 37°C. In order to detect the DIG-labelled probe, sections werepre-incubated in blocking solution (PBS containing 0.1% Tween-20 and 10%heat-inactivated goat serum) for 1 hour and then incubated over night at4° C. with a 1:5000 dilution of alkaline-phosphatase-coupled anti-DIGantibody (Boehringer Mannheim). The following day, each section wasgiven four, two-hour washes in PBS containing 0.1% Tween-20, and thengiven two ten-minute washes in NTMT buffer (100 mM NaCl, 100 mM Tris-HCl(pH9.5), 50 mM MgCl₂, 0.1% Tween-20). The sections were then incubatedin BM-purple substrate containing 0.5 mg/ml of levamisole for 48 hours.The color reaction was stopped by washing in PBS. The sections were airdried and covered with cover-slip with DPX (KEBO-lab, Sweden).

The results of the in situ hybridization reactions are presented inTable 1.

TABLE 1 Expression of neublastin in Mice Structure E13.5 E18.5 P1 P7Adult Forebrain ++ Ventral Midbrain − Dorsal Root ganglia ++ Spinalchord + Retina +++ +++ + Olfactory bulb ++ ++ ++ Tooth pulp ++ ++ +Trigeminal ganglia ++ ++ ++ Striatum + + ++ Cortex ++ ++ ++ + Dentategyrus ++ +

As shown in Table 1, at embryonic day 13.5 (“E13.5”), neublastin wasexpressed in the spinal chord and in the hindbrain, and weakly in theforebrain. Neublastin expression was also detected in the developingretina and in the sensory ganglia (dorsal root ganglia and trigeminalganglia (V)). Outside the nervous system, a weak signal was found in thekidney, the lung and the intestine, indicating that neublastin is alsoexpressed in those tissues.

At embryonic day 18.5 (“E18.5”), neublastin was expressed mostprominently in the trigeminal ganglion (V). Neublastin expression wasalso detected in the retina, the striatum, and the cortex. In addition,expression was seen in tooth anlage.

Again referring to Table 1, increased neublastin expression, from theE18.5 time-point to postnatal days 1 and 7, was seen in the cortex, thestriatum and the trigeminal ganglion (V). Neublastin expression was moreprominent in the outer layers of the cortex than in the inner layers ofthe cortex. On P7, expression was found in the same structures as at day1 but in addition neublastin expression was found in the hippocampus,especially in the dentate gyrus and in the cerebellum. In the adultmurine brain, neublastin was strongly expressed in dentate gyrus, withvery low or undetectable levels of neublastin expression detected othertissues tested.

Expression in Rat

The following experiment describes the hybridization of rat tissues witha alkaline-phosphatase-labelled oligodeoxynucleotide neublastinanti-sense probe.

Preparation of tissue samples: Rat embryos (E14) were obtained frompregnant Wistar rats (Mollegard, Denmark) following pentobarbitalanaesthesia. Postnatal rats (P0, P7, adult) were killed by decapitation.Dissected brains and whole heads were immediately immersed in cold 0.9%NaCl, fresh frozen and sectioned at 20 μm on a cryostat (coronal andsagittal sections, 10 series).

In situ hybridization: Two series of sections were hybridized using ananti-sense alkaline-phosphatase (AP) conjugated oligodeoxynucleotideprobe (5′-NCA GGT GGT CCG TGG GGG GCG CCA AGA CCG G-3′ (SEQ ID NO:27),Oligo. No. 164675, DNA Technology, Denmark,). This probe iscomplementary to bases 1140 to 1169 of the mouse neublastin cDNA of SEQID NO:15).

Prior to hybridization, the sections were air dried at room temperature,heated at 55° C. for 10 min., and then treated with 96% ethanol at 4° C.overnight. The sections were then air dried and incubated inhybridization medium (5.0 pmol probe/ml) overnight at 39° C. (Finsen etal., 1992, Neurosci. 47:105-113; West et al., 1996, J. Comp. Neurol.,370:11-22).

Post-hybridization treatment consisted of four, thirty-minute rinses in1×SSC (0.15M NaCl, 0.015 M Na-citrate) at 55° C., followed by threeten-minute rinses in Tris-HCl, pH 9.5 at room temperature prior toapplying AP developer. AP developer was prepared immediately before useand contained nitroblue tetrazoleum (NBT, Sigma), 5-bromo, 4-chloro,3-indolylphosphate (BCIP, Sigma), and Tris-HCl-MgCl₂ buffer, pH 9.5(Finsen et al., Neurosci. 1992 47 105-113). AP development took place inthe dark at room temperature for 48 hours. The color reaction wasstopped by rinsing the sections in destined water. The sections weredehydrated in graded acetone, softened in xylene-phenol creosote(Allchem, UK), cleared in xylene, and coverslipped using Eukitt (Bie &Berntsen, Denmark).

Control reactions consisted of (1) pre-treating the sections with RNaseA (50 μg/ml, Pharmacia, Sweden) prior to hybridization; (2) hybridizingthe sections with a hundred-fold excess of unlabelled probe; and (3)hybridizing the sections with hybridization buffer alone. The results ofthe hybridization reactions are presented in Table 2.

TABLE 2 Expression of neublastin in rats Structure E14 P0/P1 P7 AdultForebrain ++ Ventral Midbrain − Dorsal root ganglia ++ Spinal cord +Retina + Olfactory bulb (+) ++ ++ Cerebellum + ++ + Trigeminal ganglia++ ++ Striatum + +(+) Cortex (+) ++ ++ + Hippocampus (+) ++ ++

At embryonic day 14 (E14), neublastin was weakly expressed in ratembryos in the forebrain, in the hindbrain, and in the spinal cord.Neublastin mRNA was also detected in the eye (retina), dorsal rootganglia, the trigeminal ganglia (V), and in the kidneys, lungs, heart,liver, and intestines. In newborn (P0) rats there was marked neublastinexpression in the cortex and in the striatum. Neublastin expression wasalso detected in the olfactory bulb and in the hippocampus. In 7-day-old(P7) rats, neublastin was expressed in the cortex, the striatum, theolfactory bulb, and in the cerebellum. A marked signal was seen in thehippocampus. In adult rats, very low or undetectable levels ofneublastin expression were detected in most areas of the brain. Weaksignals were detected in the thalamic nucleus, and marked neublastinexpression was detected in the hippocampus.

Example 4

Neublastin Polypeptides

The open reading frame, or coding region (CDS), identified in SEQ ID NO:8 encodes the pre-pro-polypeptide (designated “pre-pro-neublastin”). Theamino acid sequence predicted from this open reading frame is shown inSEQ ID NO: 9. Based on SEQ ID NO: 9, three variants of neublastinpolypeptides were identified. These variants include: (i) thepolypeptide designated herein as NBN140, which possesses the amino acidsequence designated as SEQ ID NO: 10; (ii) the polypeptide designatedherein as NBN116, which possesses the amino acid sequence designated asSEQ ID NO: 11; and (iii) the polypeptide designated herein as NBN113,which possesses the amino acid sequence designated as SEQ ID NO: 12.

Similarly, based on the coding region (CDS) as identified in SEQ ID NO:3, which encodes the pre-pro-polypeptide possessing the amino acidsequence (designated as SEQ ID NO: 4), three variants of neublastin wereidentified. These variants include: (i) the polypeptide which possessesthe amino acid sequence designated as SEQ ID NO: 5; (ii) the polypeptidewhich possesses the amino acid sequence designated as SEQ ID NO: 6; and(iii) the polypeptide which possesses the amino acid sequence designatedas SEQ ID NO: 7.

Based on a Clustal W (1.75)-based multiple sequence alignment, SEQ IDNO: 9 was aligned with the amino acid sequences of GDNF, persephin andneurturin. This alignment is illustrated in Table 3.

TABLE 3 Amino Acid Sequence Comparison of Neublastin (SEQ ID NO:35) toPersephin (SEQ ID NO:36), Neurturin (SEQ ID NO:34), and GDNF (SEQ IDNO:37) Neurturin-full--------------------MQRWKAAALASVLCSSVLSIWMCREGLLLSHRLGPA NeublastinMELGLGGLSTLSHCPWPRRQPALWPTLAALALLSSVAEASLGSAPRSPAPREGPPP Persephin-full-------------------------------------------------------- GDNF_HUMAN-full-----MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDS Neurturin-fullLVPLHRLPRTLDARIARLAQYRALLQGAPDAMELRELTPWADRPPGPRRRAGPRRR NeublastinVLASPAGHLPGGRTARWCSGRARRPPPQPSRPAPPPPAPPSALPRGGRAARAGGPG Persephin-full-MAVGKFLLGSLLLLSLQLGQGWGPDARGVPVADGEFSSEQVAKAGGTWLGTHRPL GDNF_HUMAN-fullNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKG Neurturin-fullRARARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEA-AARVYDLGLRR NeublastinSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCRR-ARSPHDLSLAS Persephin-fullARLRRALSGPCQLWSLTLSVAELGLGYASEEKVIFRYCAGSCPRGARTQHGLALAR GDNF_HUMAN-fullRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDA-AETTYDKILKN          * *    : *  ****: :.* : **:*:*:*   *   :.  * Neurturin-fullLRQRRRLRRE---RVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSARECACV- NeublastinLLGAGALRPPPGSRPVSQPCCRPTRYE-AVSFMDVNSTWRTVDRLSATACGCLG Persephin-fullLQGQGRAHGG--------PCCRPTRYT-DVAFLDDRHRWQRLPQLSAAACGCGG GDNF_HUMAN-fullLSRNRRLVSD----KVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI-*                 .****  :   ::*:* .  :: : . **  *.* *indicatespositions which have a single, fully conserved residue. :indicates thatone of the following ‘strong’ groups is fully conserved: -STA, NEQK,NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW. .indicates that one of thefollowing ‘weaker’ groups is fully conserved: -CSA, ATV, SAG, STNK,STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.

From the amino acid sequence alignment shown in Table 3, it can be seenthat neublastin has seven conserved cysteine residues at locations thatare conserved within the TGF-β superfamily. In one embodiment, thepreferred neuroblastin polypeptide contains (seven) cysteines conservedas in SEQ ID NO: 2 at positions 8, 35, 39, 72, 73, 101 and 103, or as inSEQ ID NOS: 4 and 9 at positions 43, 70, 74, 107, 108, 136 and 138.These seven conserved cysteine residues are known within the TGF-bsuperfamily to form three intramonomeric disulfide bonds (contemplated,e.g., in SEQ ID NO: 2 between cysteine residues 8-73, 35-101, and39-103, and, e.g., in SEQ ID NOS: 4 and 9 between cysteine residues43-108, 70-136, and 74-138) and one intermonomeric disulfide bond(contemplated, e.g., in SEQ ID NO: 2 between cysteine residues 72-72,and, e.g., in SEQ ID NOS: 4 and 9 between cysteine residues 107-107),which together with the extended beta strand region constitutes theconserved structural motif for the TGF-b superfamily. See, e.g., Daopinet al., Proteins 1993 17 176-192.

Based on this sequence alignment, neublastin was shown to be a member ofthe GDNF subfamily of neurotrophic factors(LGLG−FR(Y/F)CSGSC−QxCCRP−SAxxCGC, the GDNF subfamily fingerprint,underlined in Table 3).

The homology of neublastin to other members of the GDNF family wascalculated, and the results are presented Table 4, below.

TABLE 4 Homology of Neublastin Polypeptides to other members of the GDNFFamily Mature Protein NBN140 Mature Protein NBN113 Homology Homology offull of full Homology length Homology length Neurotrophic Overlap Strongpeptides Overlap Strong peptides Factor Identity (aa) Homology IdentityIdentity (aa) Homology Identity GDNF 34% 137 48% 31.9% 36% 111 52% 29.5%(47/137) (67/137) (41/111) (59/111) NTN 48% 127 56% 36.9% 49% 114 57%44.7% (61/127) (72/127) (56/114) (66/114) PSP 44% 125 56% 36.9  45% 11157% 44.3% (55/125) (71/125) (51/111) (65/111) IHA 31%  81 — 25.2% 31% 81  — 22.5% (25/81)  (25/81)  TGF-β2 23%  73 — 18.5% 23%  73  — 20.2%(17/73)  (17/73)  GDNF = Glial cell line Derived Neurotrophic Factor NTN= Neurturin PSP = Persephin IHA = Inhibin-α TF-β2 = Transforming GrowthFactor-β2 Strong homology indicates that one of the following “strong”groups are conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

Example 5

Production of Neublastin

We have produced neublastin in both eukaryotic and prokaryotic cells, asdescribed below. Expression Vectors The full length cDNA encodingneublastin was inserted into the eukaryotic expression vector pUbi1Z.This vector was generated by cloning the human UbC promoter into amodified version of pcDNA3.1/Zeo. The unmodified pcDNA3.1/Zeo iscommercially available (Invitrogen). The modified pcDNA3.1/Zeo issmaller than the parent vector, because the ampicillin gene (fromposition 3933 to 5015) and a sequence from position 2838 to 3134 wereremoved. In this modified version of pcDNA3.1/Zeo, the CMV promoter wasreplaced with the UbC promoter from pTEJ-8 (Johansen et al., FEBS Lett.1990 267 289-294), resulting in pUbi1Z. Mammalian Cell Expression ThepUbi1Z vector which contained neublastin coding sequences was thentransfected into the mammalian cell line HiB5, which is an immortalisedrat neural cell line (Renfranz et al., Cell, 66, pp. 713-729 (1991)).Several HiB5 cell lines stably expressing neublastin (as determined byRT-PCR) have been established. In one of these stable cell lines,HiB5pUbi1zNBN22 expression was confirmed by hybridizing total RNA on aNorthern blot with a ³²P-labelled neublastin probe. The results of thesestudies are shown in FIG. 2. HiB5pUbi1zNBN22 was then used as a sourceof neublastin for studies of neublastin neurotrophic activity.

FIG. 2 shows the expression of neublastin cDNA in the HiB5pUbi1zNBN22clone (i.e., Northern blot probed with ³²P-labelled neublastin cDNA ofthe present invention as described infra). The blot was prepared bytotal RNA extracted from untransfected HiB5 cells, HiB5pUbi1zNBN22 cellsand HiB5pUbi1zGDNF14, respectively, as indicated. The positions of the28S and 18S rRNA bands corresponding to 4.1 kb and 1.9 kb, respectively,are indicated on the blot.

As shown in FIG. 3, antibodies raised against neublastin-derivedpolypeptides also recognised a protein of approximately 13 kilodaltons(“kD”) in conditioned medium from the HiB5pUbi1zNBN22 clone but not fromnon-transfected HiB5 cells (cf. Example 6).

The predicted molecular weights of the non-modified (i.e. lackingpost-translational modifications) neublastin polypeptides NBN140 (SEQ IDNO: 10), NBN116 (SEQ ID NO:11) and NBN113 (SEQ ID NO: 12) weredetermined to be 14.7 kilodaltons (“kD”), 12.4 kD, and 12.1 kD,respectively.

Methods: A Northern blot with total RNA (10 μg) from untransfected HiB5cells and the HiB5pUbi1zNBN22 clone was prepared by electrophoresis on a0.8% formaldehyde agarose gel and blotted onto a nylon membrane(Duralone, Stratagene). The blot was hybridized and washed as describedin Example 3 with a 1.3 kb ³²P-labelled probe prepared by randomlabelling covering SEQ ID. NO: 8 and additional nucleotides from the5′UTR and 3′UTR of the neublastin cDNA. The blot was exposed to aHyperfilm MP (Amersham) at −80° C. using intensifying screens.

Conditioned medium from Hib5pUbi1zNBN22, or untransfected Hib5 cellsincubated overnight in serum-free medium supplemented with N2 supplement(Life Technologies; Cat No. 17502-048) was concentrated and separated on15% polyacrylamide gels (Amersham Pharmacia Biotech; Cat. No.80-1262-01). Proteins were transferred to PVDF-membranes (AmershamPharmacia Biotech; Cat. No. RPN-303F) and non-specific protein-bindingsites were blocked with 5% non-fat dry milk in PBS with 0.1% Tween-20.Membranes were incubated overnight with a polyclonal neublastin antibody(1:1000), followed by incubation with a secondary anti-rabbit IgGantibody (Amersham Pharmacia Biotech; Cat. No. NA 934) conjugated tohorseradish peroxidase (1:2000). Immunostaining was visualised usingenhanced chemoluminiscence (ECL) (Amersham Pharmacia Biotech; Cat. No.RPN2109) or ECL+ (Amersham Pharmacia Biotech; Cat. No. RPN2132)according to the manufacturer's instructions (Amersham).

The results of these experiments are shown in FIG. 3. FIGS. 3A and 3Bare illustrations of the expression of neublastin protein in transfectedHiB5 cells. Overnight medium from non-transfected HiB5 cells [Lane 1],or from an HiB5 clone stable transfected with neublastin cDNA [Lane 2],were concentrated as described infra. The medium was then analyzed byWestern blotting using two different polyclonal antibodies, Ab-1 andAb-2 described in Example 10, specific for neublastin. In the mediumderived from transfected cells, both of the antibodies were found torecognize a protein with a molecular weight of approximately 15 kDa.This protein was not seen in non-transfected HiB5 cells.

The cloned cDNA encoding neublastin can also be inserted into othereukaryotic expression vector, e.g., the eukaryotic expression vectorTEJ-8 (Johansen et al., FEBS Lett., 1990, 267:289-294) or pcDNA-3(Invitrogen), and the resulting expression plasmid transfected into analternative mammalian cell line, e.g., Chinese Hamster Ovary (“CHO”)cells, the HEK293, the COS, the PC12, or the RN33b cell lines, or ahuman neural stem cell. Stable cell lines expressing neublastin areused, e.g., to produce the neublastin protein.

Expression in CHO Cells

Construction of plasmid pJC070.14 In order to express the NeublastincDNA in Chinese hamster ovary cells, a cDNA fragment encoding the preproform of human Neublastin was inserted into the mammalian expressionvector pEAG347 to generate plasmid pJC070.14. pEAG347 contains tandemSV40 early and adenovirus major late promoters (derived from plasmidpAD2beta; Norton and Coffin, Mol. Cell. Biol. 5: 281 (1985)), a uniqueNotI cloning site, followed by SV40 late transcription termination andpolyA signals (derived from plasmid pCMVbeta; MacGregor and Caskey,Nucl. Acids. Res. 17: 2365 (1989)). In addition, pEAG347 contains apUC19-derived plasmid backbone and a pSV2dhfr-derived dhfr for MTXselection and amplification in transfected CHO cells.

Plasmid pJC070.14 was generated in two steps. First, a fragment encodingthe prepro form of human Neublastin was isolated from plasmid pUbi1Z-NBNusing the polymerase chain reaction with oligonucleotides KD2-8245′AAGGAAAAAA GCGGCCGCCA TGGAACTTGG ACTTGGAGG3′ (SEQ. ID. NO. 31),KD2-825 5′TTTTTTCCTT GGCGGCCGCT CAGCCCAGGC AGCCGCAGG3′ (SEQ. ID. NO. 32)and PFU polymerase. The fragment was cloned into the Srf-1 site ofpPCR-Script Amp SK(+) to generate the plasmid pJC069. In the secondstep, a partial Not-1 digest was performed on plasmid pJC069 to generatea 685 bp fragment (containing the neublastin gene) which was cloned intothe Not-1 site of plasmid pEAG347 to generate plasmid pJC070.14.Transcription of the neublastin gene in plasmid pJC070.14 is controlledby the adenovirus major late promoter.

Generation of CHO cell lines expressing Neublastin. 200 μg of pJC070.14was linearized by digestion with the restriction endonuclease Mlu-1. TheDNA was extracted with phenol:chloroform:isoamyl alchohol (25:24:1) andethanol precipitated. The linearized DNA was resuspended in 20 mM HepespH7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM dextrose (HEBS) andintroduced into ˜4E7 CHO dukx B1(dhfr−) cells (p23) by electroporation(280V and 960 μF). Following electroporation, the cells were returned toculture in α+Modified Eagle's Medium (MEM) supplemented with 10% fetalbovine serum (FBS) for two days. The cells were then trypsinized andreplated in 100 mm dishes (100,000 cells/plate) in α-MEM (lacking ribo-and deoxyribonucleosides), supplemented with 10% dialyzed FBS, for fivedays. The cells were subsequently split at a density of 100,000cells/100 mm plate, and selected in 200 nM methotrexate. Resistantcolonies were picked and scaled up to 6 well plates; conditioned mediafrom each clone was screened using a specific assay for neublastindescribed below. The twelve clones expressing the highest level ofneublastin were scaled up to T162 flasks and subsequently reassayed. Asshown in FIG. 10, the CHO cell lines produced neublastin in the range of25 to 50 ng/ml.

Ternary complex assay for neublastin. We assayed for the presence ofneublastin in the media of CHO cell line supernatants using a modifiedform of a ternary complex assay described by Sanicola et al. (Proc NatlAcad Sci USA 94: 6238 (1997).

In this assay, the ability of GDNF-like molecules can be evaluated fortheir ability to mediate binding between the extracellular domain of RETand the various co-receptors, GFRα1, GFRα2, and GFRα3. Soluble forms ofRET and the co-receptors were generated as fusion proteins. A fusionprotein between the extracellular domain of rat RET and placentalalkaline phosphatase (RET-AP) and a fusion protein between theextracellular domain of rat GFRα1 (disclosed in published applicationWO9744356; Nov. 27, 1997, herein incorporated by reference) and the Fcdomain of human IgG1 (rGFRα1-Ig) have been described (Sanicola et al.Proc Natl Acad Sci USA 94: 6238 (1997)).

To generate a fusion protein between the extracellular domain of murineGFRα3 and the Fc domain of human IgG1 (mGFRα3-Ig), a DNA fragmentencoding amino acids 1-359 of murine RETL3 was ligated to a fragmentcontaining the Fc domain of human IgG1 and cloned into the expressionvector pEAG347 to generate plasmid pGJ144. Plasmid pGJ144 wastransfected into Chinese hamster ovary cells (CHO) to generate a stablecell line producing the fusion protein, which was purified on a ProteinA Sepharose immunoaffinity column using standard methods. In summary, ifthe GDNF-like molecule can mediate binding of the co-receptor to RET inthis assay, then the RET-AP fusion protein will be retained on the plateand the amount that is retained can be measured using a chemiluminescentsubstrate for alkaline phosphatase.

Dynex Microlite-1 ELISA plates (Dynex Technologies) were coated with 1μg/ml goat antibody specific for human Fc in 50 mMbicarbonate/carbonate, pH 9.6 for 16 hr. The plates were emptied andfilled with 300 μl of 1% I-block (Tropix) in TBS/0.5% Tween-20 (TBST),for 1 hr. After washing three times with TBST the wells were filled with100 μl of 1 μg/ml RGFRα1-Ig or mGFRα3-Ig diluted in conditioned mediafrom 293 EBNA cells expressing the RET-AP fusion gene. 100 ul ofconditioned media from the CHO neublastin clones was then added to thetop well of a column of wells, and 2 fold serial dilutions wereperformed down each row of wells, and incubated for 1.5 hr at roomtemperature. The plates were then washed three times with TBST, andtwice with 200 mMTris pH9.8, 10 mMMgCl₂ (CSPD buffer). The wash solutionwas then replaced with 425 μM CSPD (Tropix) in CSPD buffer containing 1mg/ml Sapphire chemiluminescence enhancer (Tropix), and incubated for30′ at room temperature. The chemiluminescent output was measured usinga Dynatech luminometer.

In the initial experiments, we investigated whether neublastin producedby the CHO cell lines could mediate the binding of GFRα1 or GFRα3 to theextracellular domain of RET. As shown in FIG. 11, conditioned mediumfrom CHO cell clone #53 produced a robust signal in the ternary complexassay when the mGFRα3-Ig fusion protein was included, but no signal whenthe rGFRα1-Ig fusion protein was included, indicating that neublastinbinds to GFRα3 but not to GFRα1. This behavior clearly distinguishesneublastin from GDNF; as shown in FIG. 11, GDNF binds to GFRα1 but notto GFRα3. No signal was observed with either co-receptor fusion protein,when conditioned medium from the parental CHO cell line or straightmedium was assayed.

In order to quantitate the expression levels of neublastin in the CHOcell lines, a standard curve was prepared using rGFRα1-Ig and GDNFstarting at a concentration of 1 ng/ml. Neublastin concentrations forthe different CHO cell lines were then calculated using this standardcurve; the levels produced by five CHO cell lines are shown in FIG. 10.Because this estimation depends on the untested assumption that thebinding affinity between GDNF and GFRα1 is similar to the bindingaffinity between neublastin and GFRα3, these levels are onlyapproximate.

Analysis of neublastin from CHO cell line supernatants. In order tofurther analyze the neublastin produced by the CHO cell lines, theprotein was extracted from the medium using the GFRα3-Ig fusion proteinand analyzed by western blots with polyclonal antibodies raised againstneublastin peptides.

In the first experiment, the neublastin was extracted with mGFRα3-Igattached to Sepharose beads. mGFRα3-Ig was attached to Sepharose beadsusing the conditions suggested by the manufacturer, Pharmacia Inc. 100μL of mGRFα3-Ig-Sepharose was added to 1.0 mL samples of conditionedmedium from a negative control CHO cell line or from the neublastinproducing CHO cell line #16. The suspensions were incubated for twohours on a rocking platform. Each suspension was centrifuged and thesupernatant removed followed with three 1.0 mL washes with 10 mM HEPES,100 mM NaCl, pH 7.5. Each resin was resuspended in 100 μL of 2× reducingsample buffer and heated to 100° C. for 5 minutes. 20 μL of the samplebuffer supernatant and 10 uL of a molecular weight standard (FMC) wereapplied to each well of a 10-20% precast SDS-PAGE gel (Owl Scientific).The gel was electrophoresed at 40 mA constant current for 72 minutes.

For western blot analysis, the protein was electroblotted tonitrocellulose (Schleicher and Schuell) in a Hofer Scientific apparatusin 10 mM CAPS, 10% methanol, 0.05% SDS, pH 11.2 buffer system (45minutes at 400 mA constant current). After the transfer, thenitrocellulose filter was removed from the cassette and the molecularweight markers were visualized by staining with a solution of 0.1%Ponceau S in 1% acetic acid for 60 seconds. The membrane was cut intotwo sections and the excess stain was removed by gentle agitation indistilled water. The membranes were blocked in 2% nonfat dry milk in TBSovernight at 4° C. The membranes were incubated individually with two ofthe affinity-purified anti-neublastin peptide antibodies (R30 and R31)at a concentration of 1.0 μg/mL in 2% nonfat dry milk in TBS). Themembranes were washed with three 10 minute washes in TBS-Tween andincubated in a 1:5000 dilution of goat anti-rabbit IgG-HRP conjugate(Biorad) for 30 minutes. The membranes were washed with three 10 minutewashes of TBS-Tween and developed with ECL substrate (Amersham). Asshown in FIG. 12, specific bands were detected in the proteins extractedfrom the neublastin producing CHO cell line with both antibodies (lanes2 and 4), when compared to the bands observed in the extracted proteinsfrom the negative control cell line (lanes 1 and 3).

The molecular weight of the lower species is about 13 kD and probablyrepresents the mature domain of neublastin, generated after cleavage ofthe pro-domain. This cleavage could occur after any one of the threeArg-_ (e.g., -RXXR↓-) residues of the prepro neublastin protein togenerate either the 140 AA, 116 AA or 113 AA forms, as set forth inSEQ.ID.NOS. 10, 11, or 12, respectively. The predicted molecular weightsof the non-modified (i.e., lacking post-translational modifications)neublastin polypeptides NBN140 (SEQ. ID. NO. 10), NBN116 (SEQ. ID. NO.11), and NBN113 (SEQ. ID. NO. 12) were determined to be 14.7 kD, 12.4kD, and 12.1 kD, respectively. Further analysis will be needed toconfirm the structure of this species as well as the other neublastinspecific bands.

In the second experiment, the neublastin was extracted with hGFRα3-Igcaptured on an ELISA plate. To generate a fusion protein between theextracellular domain of human GFRα3 (disclosed in published applicationWO97/44356; Nov. 27, 1997, herein incorporated by reference) and the Fcdomain of human IgG1 (hGFRα3-Ig), a DNA fragment encoding amino acids1-364 of human GFRα3 was ligated to a fragment containing the Fc domainof human IgG1 and cloned into the expression vector CH269 described bySanicola et al. (Proc Natl Acad Sci USA 94: 6238 (1997)). The fusionprotein encoded by this plasmid was transiently expressed in293-Epstein-Barr virus-encoded nuclear antigen (EBNA) cells and purifiedon a Protein A Sepharose immunoaffinity column using standard methods.

Six wells of a 96-well plate were coated overnight at 4° C. with goatanti-human IgG (Fcγ fragment specific; Jackson immunulogics) at aconcentration of 25 μg/ml in PBS (300 μl/well). The wells were blockedfor 1 h at room temperature with 400 μl of 1% BSA in PBS. After 3 washeswith PBST (PBS+0.05% Tween 20), 300 μl hGFRα3-Ig (10 μg/ml in PBScontaining 0.1% BSA) was added to each well. The plate was incubated for1 h at room temperature and shaken gently (200 strokes/min) to maximizethe binding. The wells were then emptied and washed again 3 times withPBST. 250 μl of conditioned media from a negative control CHO cell lineor from the neublastin producing CHO cell line #25 was added to each of3 wells. The plate was incubated for 3 h at room temperature and shakengently (300 strokes/min). The wells were then washed twice with PBST. 25μl of non-reducing Laemli loading buffer was added to the first well andthe plate was shaken rapidly for 5 min to elute the bound proteins (1300strokes/min). The content was transferred to the next well and theprocedure was repeated to elute the proteins bound in the second andthird wells. After adding β-mercaptoethanol (5% final), the samples wereboiled for 5 minutes and analyzed by SDS-PAGE on a 10-20% polyacrylamidegel.

For western blot analysis, the proteins were transferred tonitrocellulose. The membrane was blocked and probed in 5% non fat drymilk, PBST and washed in PBST. Neublastin was detected byelectrochemoluminescence after reaction with polyclonal antibodies (R30and R31) raised against two neublastin peptides (at 1 ug/ml) followed byreaction with HRP-conjugated goat anti-rabbit antibodies (BioRad). Asshown in FIG. 13, five neublastin specific bands were detected in theextracted proteins from the neublastin producing CHO cell line (lane 2).The lower two bands are very similar to the bands observed in FIG. 12;again, the lower band probably represents the mature domain ofneublastin generated after cleavage of the pro-domain.

Subsequent analysis (data not shown) of the bands in FIG. 13 shows thatdeglycosylation with PGNase F of the approximately 18 kD band reducesthat band to a size equivalent to the lower-most band in the gel of FIG.13. This suggests that neublastin may be produced as a glycosylatedprotein in mammalian cells.

Expression of Neublastin in E. coli

In order to express the neublastin gene in E. coli, syngenes wereconstructed with lower GC content and preferred E. coli codons. Thesyngene is being cloned into two vectors, pET19b and pMJB164, aderivative of pET19b. The construction with pET19b is shown in FIG. 14.In this construct, the sequence encoding the mature domain of neublastin(NBN113) is directly fused to an initiating methionine. The constructionwith pMJB164 is shown in FIG. 15. In this construct, the mature domainof neublastin is fused to a histidine tag (i.e. 10 histidines) separatedby an enterokinase cleavage site. The initiating methionine precedes thehistidine tag.

Nucleotide Sequence Encoding Neublastin in FIG. 14

ATGGCTGGAGGACCGGGATCTCGTGCTCGTGCAGCAGGAGCACGTGGCTGTCGTCT

GCGTTCTCAACTAGTGCCGGTGCGTGCACTCGGACTGGGACACCGTTCCGACGAACT

AGTACGTTTTCGTTTTTGTTCAGGATCTTGTCGTCGTGCACGTTCTCCGCATGATCTA

TCTCTAGCATCTCTACTAGGAGCCGGAGCACTAAGACCGCCGCCGGGATCTAGACCT

GTATCTCAACCTTGTTGTAGACCTACTAGATACGAAGCAGTATCTTTCATGGACGTA

AACTCTACATGGAGAACCGTAGATAGACTATCTGCAACCGCATGTGGCTGTCTAGGA

TGATAATAG  SEQ. ID. NO.29

Nucleotide Sequence Encoding His-tagged Neublastin in FIG. 15

ATGGGCCATCATCATCATCATCATCATCATCATCACTCGAGCGGCCATATCGACGAC

GACGACAAGGCTGGAGGACCGGGATCTCGTGCTCGTGCAGCAGGAGCACGTGGCTG

TCGTCTGCGTTCTCAACTAGTGCCGGTGCGTGCACTCGGACTGGGACACCGTTCCGA

CGAACTAGTACGTTTCGTTTTTGTTCAGGATCTTGTCGTCGTGCACGTTCTCCGCAT

GATCTATCTCTAGCATCTCTACTAGGAGCCGGAGCACTAAGACCGCCGCCGGGATCT

AGACCTGTATCTCAACCTTGTTGTAGACCTACTAGATACGAAGCAGTATCTTTCATG

GACGTAAACTCTACATGGAGAACCGTAGATAGACTATCTGCAACCGCATGTGGCTGT

CTAGGATGATAATAG  SEQ. ID. NO. 30.

Example 6

Effect of Neublastin on the Survival of Rat Embryonic DopaminergicNeurons and ChAT Activity

In this series of experiments the effect of conditioned medium fromneublastin-producing HiB5pUbi1zNBN22 cells described above was assessed.

Preparation of Cultures: The ventral mesencephalon or spinal chord wasdissected out from rat E14 embryos in cold Hanks Buffered Salt Solution(HBSS). Tissue pieces were incubate in sterile filtered 0.1% trypsin(Worthington) and 0.05% DNase (Sigma) in HBSS at 37° C. for 20 min.Tissue pieces was then rinsed four times in HBSS+0.05% DNase anddissociated using a 1 ml automatic pipette. The suspension was thencentrifuged at 600 rpm for 5 min and the pellet was re-suspended inserum conditioned medium (SCM; DMEM with 10% foetal calf serum). Thetotal number of cells was assessed by tryphan blue dye exclusion methodand plated at a density of 100.000 cells/cm² in poly-L-lysine coatedeight-well chamber slides (Nunc) for assessment of dopaminergic neuronsurvival or at 200 000 cells/cm² in 48 well plates (Nunc) for ChATactivity measurements. Cells were incubated in SCM at 5% CO₂/95% O₂ and95% humidity in 37° C. for 24-48 h before changing to serum free medium(SFM) with addition of neurotrophic factors.

Cells for assessing dopaminergic neuron survival was left for 5 days inSFM+trophic factor additions and then fixed for 5 min in 4% PFA andstained for tyrosine hydroxylase by immunohistochemistry.

Cells for ChAT activity were left for 3 days with SFM and then lysed inHBSS+0.1% Triton X-100 and immediately frozen down on dry ice until Chatactivity measurement.

Trophic Factor Addition: Conditioned medium was collected fromnon-transfected HiB5 control or HiB5 producing neublastin(HiB5pUbi1zNBN22) or GDNF (HiB5pUbi1zGDNF-L17). HiB5pUbi1zNBN22 producesapproximately 20 ng GDNF/24 hours/10⁵ cells as determined by GDNF-ELISAon conditioned medium, collected from the cells. The respective celllines were incubated overnight with DMEM+1% FCS and the supernatant wastaken off and stored at −20° C. until use. The supernatants were dilutedin 1:50 in SFM when added to the cells. Separate wells were treated withHiB5 control supernatant (1:50)+purified. recombinant rat GDNF (from0.03-10 ng/ml).

The results of these experiments are shown in FIG. 4. FIGS. 4A-4C areillustrations of the effect of neublastin, secreted from HiB5pUbi1zNBN22cells, on the survival of cultured rat embryonic, dopaminergic, ventralmesencephalic neurons and ChAT activity in cholinergic cranial nervemotor neurons in serum-free medium as described infra in Example 5.1.

FIG. 4A is an illustration of the dose-response curve for recombinantGDNF on ChAT activity (dpm/hour) measured at DIV5 in serum-free cultureswhich were initially established from E14 ventral mesencephali [i.e.,HiB5; GDNF 0.03 ng/ml; GDNF 0.1 ng/ml; GDNF 0.3 ng/ml; GDNF 1 ng/ml;GDNF 10 ng/ml; GDNF 100 ng/ml].

FIG. 4B is an illustration of ChAT activity (dpm/hour) measured at DIV5in serum-free cultures which were initially established from E14 ventralmesencephali. Diluted conditioned medium from either neublastinproducing HiB5pUbi1zNBN22 cells (neublastin) or GDNF-producingHiB5GDNFL-17 (GDNFL-17) cells were added as indicated in the figure[i.e., neublastin 1:10; neublastin 1:50; GDNF L-17 1:50].

FIG. 4C is an illustration of the number of tyrosine hydroxylaseimmunoreactive cells per well [No. TH+cells/well] at DIV7 in serum-freecultures which were initially established from E14 rat ventralmesencephali. Diluted conditioned medium from either non-transfectedHiB5 cells (HiB5) or neublastin-producing HiB5pUbi1zNBN22 cells(neublastin) or recombinant GDNF, in various concentrations, were addedas indicated in the figure [i.e., HiB5 1:10; HiB5 1:40; GDNF 0.1 ng/ml;GDNF 10 ng/ml; GDNF 100 ng/ml; and neublastin 1:40].

Conditioned medium from neublastin transfected HiB5 cells diluted 1:40significantly increases the number of TH immunoreactive cells pr. wellcompared to control (untransfected) HiB5 cells at an equivalent and alower dilution (1:10 and 1:40) (see, e.g., FIG. 4B). The increase inTH-immunoreactive cells are comparable to the increase seen at a maximalGDNF concentration (10 ng/ml). This indicates that neublastin secretedto the medium has an effect on survival of the dopaminergic neuronpopulation from rat embryonic ventral mesencephalon. In contrast, unlikeGDNF secreted from transfected HiB5 cells, no effect of conditionedmedium from neublastin transfected HiB5 cells is seen on anotherneuronal population in the same culture, the cholinergic neurons (see,e.g., FIG. 4A).

Example 7

Effect of Neublastin on the Survival of Slice Cultures of Pig EmbryonicDopaminergic Ventral Mesencephalic Neurons

In this experiment the effect of co-culturing neublastin-producingHiB5pUbi1zNBN22 cells with slice cultures of ventral mesencephali fromporcine embryos.

Preparation of Cultures: Ventral mesencephali (VM) were isolated fromporcine embryos (E28; n=12) under sterile conditions, chopped into 400μm slices and placed in chilled Gey's balanced salt solution (GIBCO)with glucose (6.5 mg/ml). The tissue slices were cultured by theinterface culture method, originally developed by Stoppini et al. [L.Stoppini, P. A. Buchs, D. Muller, J. Neurosci. Methods 1991 37 173-182].

In brief, slices were placed on semi-porous membranes (Millipore, 0.3μm; 8 slices/membrane corresponding to one VM) placed as inserts in6-well plates (Costar) with serum containing medium (Gibco BRL). Eachwell contained 1 ml medium (50% Optimem, 25% horse serum, 25% Hank'sbalanced salt solution (all GIBCO)) supplemented with D-glucose to afinal concentration of 25 mM. At day 0, 7000 transfected HiB5pUbi1zNBN22(neublastin) or 7000 non-transfected HiB5 cells (control) were seeded oneach tissue slice. The co-cultures were first grown in an incubator at33° C. for 48 hours allowing the HiB5 cells immortalized with atemperature sensitive oncogene to proliferate, and then placed in anincubator at 37° C., where the HiB5 cells differentiate. The medium waschanged twice a week. Antimitotics and antibiotics were not used at anystage.

Determination of Dopamine by HPLC: At day 12 and 21 in vitro, theculture medium was collected and analysed for dopamine using HPLC withelectrochemical detection (W. N. Slooth, J. B. P. Gramsbergen, J.Neurosci. Meth. 1995 60 141-49).

Tissue Processing and Immunohistochemistry: At day 21, the cultures werefixed in 4% paraformaldehyde in phosphate buffer for 60 min., dehydratedin a 20% sucrose solution for 24 hours, frozen, cryostat sectioned at20μm (4 series), and mounted onto gelatine coated microscope slides. Oneseries of sections was immunostained for tyrosine hydroxylase (TH).Briefly, sections were washed in 0.05M tris-buffered saline (TBS, pH7.4) containing 1% Triton X-100 for 3×15 min. and incubated with 10%fettle bovine serum (FBS, Life Technologies) in TBS for 30 min. Thetissue was then incubated for 24 hours at 4° C. with monoclonal mouseanti-TH antibody (Boehringer Mannheim) diluted 1:600 in TBS with 10%FBS. After rinsing in TBS with 1% Triton X-100 for 3×15 min., sectionswere incubated for 60 min. with bio-tinylated anti-mouse IgG antibody(Amersham) diluted 1:200 in TBS with 10% FBS. The sections were thenwashed in TBS with 1% Triton X-100 (3×15 min.) and incubated for 60 min.with streptavidin-peroxidase (Dako) diluted 1:200 in TBS with 10% FBS.After washing in TBS (3×15 min.), bound antibody was visualised bytreatment with 0.05% 3,3-diaminobenzidine (Sigma) in TBS containing0.01% H₂O₂. Finally, the sections were dehydrated in alcohol, cleared inxylene, and cover-slipped in Eukitt.

Cell counts and morphometric analysis: Quantification of immunoreactiveTH-ir neurons was performed using bright field microscopy (Olympus).Only cells displaying an intense staining with a well preserved cellularstructure and a distinct nucleus were counted. The estimation was basedon cell counts in every fourth culture section using a ×20 objective.Cell numbers were corrected for double counting according toAbercrombie's formula (M. Abercrombie, Anat. Rec. 1946 94 239-47), usingthe average diameter of the nuclei in the TH-ir neurons (6.6±0.2 μm,n=30). The size of the nuclei was estimated using a neuron tracingsystem (Neurolucida, MicroBrightField, Inc.).

The results of these experiments are shown in FIG. 5. FIGS. 5A-5C areillustrations of the effect of neublastin secreted from HiB5pUbi1zNBN22cells on the fictions and survival of slice cultures of pig embryonicdopaminergic ventral mesencephalic neurons co-cultured with eitherHiB5pUbi1zNBN22 cells (neublastin) or HiB5 cells (control) as describedinfra. FIG. 5A and FIG. 5B: illustrate dopamine released to the mediumat DIV12 [Dopamine (pmol/ml)−day 12] and DIV21 [Dopamine (pmol/ml)−day21], respectively. FIG. 5C is an illustration of the number of tyrosinehydroxylase immunoreactive cells per culture [TH-ir cells per culture]at DIV21.

At day 12 HPLC analysis revealed that medium from HiB5-neublastinco-cultures contained 84% more dopamine than medium from HiB5-Cco-cultures (FIG. 5A). At day 21 the difference was 78% (FIG. 5B), andcell counts showed that HiB5-neublastin co-cultures contained 66% moretyrosine hydroxylase immunoreactive neurons than HiB5-C co-cultures(P<0.05) (FIG. 5C). This indicates, that neublastin secreted from theHiB5pUbi1zNBN22 clone has a potent survival effect on embryonic porcinedopaminergic neurons.

Example 8

Survival of Dorsal Root Ganelion Cells in Serum-free Medium

This example shows the neurotrophic activity of a neublastin polypeptidein comparison with known neurotrophic factors.

Pregnant female mice were killed by cervical dislocation. The embryoswere processed for culture as follows.

Electrolytically sharpened tungsten needles were used to dissect dorsalroot ganglia from indicated stages of C57/B16 mice (Mollegaard Breeding,Denmark). Embryonic ganglia were incubated for 5 minutes at 37° C. with0.05% trypsin (Gibco/BRL) in calcium and magnesium-free Hanks balancedsalt solution. Postnatal ganglia were treated with collagenase/dispase 1mg/ml for 30 to 45 minutes and then trypsin/DNAse 0.25% for 15 minutes.After removal of the trypsin solution, the ganglia were washed once with10 ml of DMEM containing 10% heat inactivated horse serum, and weregently triturated with a fire-polished Pasteur pipette to give a singlecell suspension.

The cells were plated on 24 well plates (Nunc), that were precoated withpolyomithine (0.5 mg/ml, overnight) and laminin (20 mg/ml for 4 h;Gibco/BRL). The neurons were incubated at 37° C. in a humidified 5% CO₂incubator in a defined medium consisting of Hams F14 supplemented with 2mM glutamine, 0.35% bovine serum albumin, 60 ng/ml progesterone, 16mg/ml putrescine, 400 ng/ml L-thyroxine, 38 ng/ml sodium selenite, 340ng/ml triiodo-thyronine, 60 mg/ml penicillin and 100 mg/ml streptomycin.

After 48 hours of incubation, neurons were clearly recognised by theirbipolar morphology under phase-contrast optics. The percentage neuronalsurvival in the absence or presence of trophic factors (added to theculture medium prior to plating the neurons at 10 ng/ml), or ofconditioned medium from the neublastin producing HiB5pUbi1zNBN22 cells,was assessed by counting the neurons in the wells at 48 hours.

The results of these experiments are presented in FIG. 9, in whichfigure:

0 represents the control experiment (in absence of factors);

1 represents experiments in the presence of GDNF;

2 represents experiments in the presence of Neuturin;

3 represents experiments in the presence of Neublastin of the invention;

E12 represents data from experiments carried out on DRG cells isolatedfrom embryonic day 12;

E16 represents data from experiments carried out on DRG cells isolatedfrom embryonic day 16;

P0 represents data from experiments carried out on DRG cells isolatedfrom the day of birth;

P7 represents data from experiments carried out on DRG cells isolatedfrom day 7 after birth; and

P15 represents data from experiments carried out on DRG cells isolatedfrom day 15 after birth.

These results clearly show that the neurotrophic factor of the inventionshow activities comparable to, or even better than those of the wellestablished neurotrophic factors.

Example 9

In vivo Effects of Neublastin on Nigral Dopamine Neurons

In order to test the ability of neublastin (neublastin) to protect adultnigral dopamine (DA) neurons from 6-hydroxydopamine induceddegeneration, we employed a rat model of Parkinson's disease ([Sauer andOertel, Neuroscience 1994 59, 401-415) and lentiviral gene transfer ofneublastin.

Lentivirus production: To generate a lentiviral transfer vector encodingneublastin, pHR′-neublastin, a 1331 bp BamH1 fragment from neublastincDNA was subcloned in the BamH1/Bg1 II site of pSL301 (Invitrogen). Fromthis construct a 1519 bp BamH1/Xho1 fragment was cut out and ligated inthe BamH1/Xho1 site of pHR′ carrying a woodchuck hepatitis viruspost-translational fragment [Zufferey R, Donello J E, Trono D, Hope T J.Woodchuck hepatitis virus posttranscriptional regulatory elementenhances expression of transgenes delivered by retroviral vectors”; J.Virol. 1999 73 (4) 2886-2892]. To generate pHR-GDNF a 701 bp BamH1/Xho1fragment from pUbi1z-GDNF was ligated in the BamH1/Xho1 site of pHR′.

Production of the lentiviral vector have been described by e.g. Zuffereyet al. [Zufferey R, Nagy D, Mandel R J, Naldini L, Trono D: “Multiplyattenuated lentiviral vector achieves efficient gene delivery in vivo;Nat. Biotechnol. 1997 15 (9) 871-875]. Briefly, the transfer constructsand the helper plasmids pR8.91 and pMDG were co-transfected into 293Tcells. Virions released into the media were collected at 48 and 72 hrspost-transfection. To concentrate the virus, the media was centrifuged1.5 hrs at 141 000 g, and the pellet dissolved in DMEM. The titer of acontrol carrying the gene for Green Fluorescent Protein (“GFP”) wasdetermined to be 10⁸ transforming units (TU)/ml by GFP fluorescence in293T cells. A RNA slot blot technique [von Schwedler U. Song J, Aiken C,Trono D: “Vif is crucial for human immunodeficiency virus type 1proviral DNA synthesis in infected cells”; J. Virol. 1993 67 (8)4945-4955] was used to determine viral particle titer. In the GDNFsupernatant and neublastin supematant there was 10 times less particlesas compared to the GFP supematant.

Surgical Procedures: All work involving animals was conducted accordingto the rules set by the Ethical Committee for Use of Laboratory Animalsat Lund University.

A total of 21 young adult female Sprague-Dawley rats (B&K Universal,Stockholm, Sweden) were used and housed under 12 hours light:dark cyclewith free access to rat chow and water. Retrograde labelling and 6-OHDAlesions were performed 3 weeks prior to lesion according to Sauer andOertel [Sauer and Oertel, Neuroscience 1994 59:401-415]. Briefly, underEquithesin anaesthesia (0.3 ml/100 g) the rats were injected bilaterallywith 0.2 μl of a 2% solution (dissolved in 0.9% NaCl) of the retrogradetracer Fluoro-Gold (FG; Fluorochrome, Inc., Englewood, Colo.).Injections were made using a 2 μl Hamilton syringe at co-ordinates:AP=+0.5 mm; ML=±3.4 mm relative to bregma; DV=−5.0 mm relative to thedura and incisor bar set to 0.0 mm. In addition, 0.05 μl/min wasinjected with another 5 min left before the needle was retracted.

Fourteen days after the FG injections animals received a total of 5deposits (1 μl/deposit) of a lentiviral vector carrying the gene forgreen fluorescent protein (GFP), neublastin or GDNF. Four of thedeposits were into the striatum along two needle tracts at the followingco-ordinates: AP=+1.0 mm, ML=−2.6 mm, DV₁−5.0 mm DV₂=−4.5 mm and AP=0.0mm, ML=−3.7 mm, DV₁=−5 mm DV₂=−4.5 mm. The supranigral deposit was madeat AP=−5.2 mm, ML=−2.0 mm, DV₁=−6.3 mm. Tooth bar was set at −2.3 mm.

Twenty-one days after retrograde labelling, and 7 days after lentiviralinjections the animals were re-anaesthetised and with a 10 μl Hamiltonsyringe a single deposit of 20kg 6-OHDA (Sigma; calculated as free baseand dissolved in 3 μl ice cold saline supplemented with 0.02% ascorbicacid) was injected into the right striatum in the same location as theFG deposits. The injection rate was 1 μl/min, leaving another 3 minbefore retracting the needle.

Tissue Processing: At 21 days after the 6-OHDA injection the animalswere deeply anaesthetised with chloral hydrate and transcardiallyperfused with saline (pH 7.4; room temperature) for one min followed by200 ml ice cold formaldehyde solution (4% paraformaldehyde in 0.1Mphosphate buffer, pH 7.4). The brains were dissected and postfixed inthe same fixative for 3-4 hours and then transferred into 25%sucrose/0.1M phosphate buffer for 48 hours. Five series of 40 μmsections through the striatum and substantia nigra (SN) were cut on afreezing microtome.

Quantitative Assessment of Dopaminergic Neurons in the SN: The number ofFG-labelled in the SN pars compacta was assessed by a blinded observeras described previously [Sauer and Oertel, Neuroscience 1994 59,401-415]. In brief, three consecutive sections centred around the levelof the medial terminal nucleus of the accessory optic tract (MTN; −5.3in the atlas of Paxinos and Watson (1997)) were used and alllabelled/stained neurons laterally to the MTN was counted at40×magnification (n=6-7/group). FG-labelled neurons were included ifthey were brightly fluorescent under epi-illumination at 330 nm,displayed a neuronal profile and extend at least one neuritic process.

On the lesion side in animals receiving injections of lentiviruscarrying GFP the number of FG-positive nigral neurons were reduced to18% of that on the intact side. In contrast, animals injected withlenti-neublastin showed a near complete protection of the number ofFG-positive nigral neurons (89%). This was as efficient as lenti-GDNFtreated animals where 87% of the retrogradely labelled neurons remainedon the lesioned side. This shows that neublastin is a potent survivalfactor for lesioned adult nigral dopamine neurons and that it is aspotent as GDNF.

FIG. 6 is an illustration of the in vivo effect of lentiviral-producedneublastin on nigral dopamine neurons. Neurons of the SN pars compacta,in female Sprague Dawley rats, were retrogradely-labelled withFluorogold (FG), 3 weeks prior to a single injection of6-hydroxydopamine (6-OHDA) in the right striatum. One week before the6-OHDA injection, the animals received injections with lentiviralvectors expressing neublastin [neublastin], GDNF [GDNF] or the GreenFluorescent Protein [GFP] as indicated in the figure. Twenty one daysafter the 6-OHDA injections, the number of FG-labelled neurons in bothsides of the striata were determined. The figure shows the percentage[%FG lesion/intact] of FG-labelled neurons in the lesioned (right) sideverses the intact (left) side of the striata of the three groups ofanimals.

Example 10

Production of Antibodies

To prepare antibodies against neublastin, two rabbits were immunisedwith either peptide 1: CRPTRYEAVSFMDVNST (amino acids 108-124 of SEQ IDNO: 9); or peptide 2: ALRPPPGSRPVSQPC (amino acids 93-107 of SEQ ID NO:9) conjugated to carrier protein at 3 week intervals. Two rabbits foreach peptide were immunized at week 0, 3, 6 and 10, and bleeds werecollected at week 7 and 11. The second bleed was affinity purified via apeptide affinity column. The antibodies were named Ab-1 and Ab-2,according to the peptide. Western blot: 2×10⁶ HiB5 cells, stablytransfected with the cDNA for neublastin (Hib5pUbi1zNBN22), oruntransfected HiB5 cells, were incubated overnight in serum free mediumwith N₂ supplement (GIBCO). The medium was concentrated on smallconcentrators with cut-off membranes of 5 kDa (Millipore, Bedford,Mass.). Concentrated samples were added 5×Laemmli sample buffer and wereheated to 95° C. for 5 minutes. Samples were separated by SDSpolyacrylamide gel electrophoresis on 15% acrylamide gels andtransferred to PVDF-membranes. Residual protein-binding sites wereblocked with 5% non-fat dry milk in PBS with 0.1% Tween-20. Membraneswere incubated overnight with neublastin antibody (1:1000), followed byincubation with a secondary anti-rabbit or anti-mouse IgG antibodyconjugated to horseradish peroxidase (1:2000).

Immunostaining was visualized using enhanced chemoluminiscence Plus(ECL+) according to the manufacturer's instructions (Amersham). Theresults of these experiments are shown in FIG. 3 and Example 5.

Using standard techniques, we also raised rabbit polyclonal antibodiesagainst the following peptides:

Peptide R27: GPGSRARAAGARGC (amino acids 30-43 of SEQ ID NO:9);

Peptide R28: LGHRSDELVRFRFC (amino acids 57-70 of SEQ ID NO:9);

Peptide R29: CRRARSPHDLSL (amino acids 74-85 of SEQ ID NO:9);

Peptide R30: LRPPPGSRPVSQPC (amino acids 94-107 of SEQ ID NO:9); and

Peptide R31: STWRTVDRLSATAC (amino acids 123-136 of SEQ ID NO:9).

Only peptides R30 and R31, relatively close to the C-terminus,recognized the denatured protein under reducing conditions on a Westernblot.

Description of Sequences Contained in the Sequence Listing SEQ ID NO.: 1Human neublastin nucleic acid.  865 bp SEQ ID NO.: 2 Human neublastinpolypeptide from sequence 1.  200 aa SEQ ID NO.: 3 Coding region (CDS)of a human pre-pro- polypeptide.  861 bp SEQ ID NO.: 4 Human neublastinpolypeptide from sequence 3.  238 aa SEQ ID NO.: 5 Variant of humanneublastin in sequence 4 (Xaa is Asn or Thr;  140 aa Yaa is Ala or Pro).SEQ ID NO.: 6 Variant of human neublastin in sequence 4 (Xaa is Asn orThr;  116 aa Yaa is Ala or Pro). SEQ ID NO.: 7 Variant of humanneublastin in sequence 4 (Xaa is Asn or Thr;  113 aa Yaa is Ala or Pro).SEQ ID NO.: 8 cDNA from positive colony PCR of human fetal brain cDNA. 861 bp SEQ ID NO.: 9 human fetal brain pre-pro-neublastin polypeptideincluding “stop”  221 aa (corresponds to seq. 8) SEQ ID NO.: 10 Variantof pre-pro-neublastin (seq. 9) NBN14O, 14.7 kD.  140 aa SEQ ID NO.: 11Variant of pre-pro-neublastin (seq. 9) NBN116, 12.4 kD.  116 aa SEQ IDNO.: 12 Variant of pre-pro-neublastin (seq. 9) NBN113, 12.1 kD.  113 aaSEQ ID NO.: 13 PCR product from screen of human fetal brain cDNA masterplate  102 bp using SEQ. ID. NOS. 17 and 18 as primers. SEQ ID NO.: 14PCR product from screen of mouse fetal cDNA master plate  220 bp usingSEQ. ID. NOS. 21 and 22 as primers. SEQ ID NO.: 15 Full length mouseneublastin cDNA. 2136 bp SEQ ID NO.: 16 Mouse pre-pro-neublastinpolypeptide.  224 aa SEQ ID NO.: 17 “NBNint.sence” Top Primer for NBNfrom human fetal brain cDNA  18 nt complementary to bases 551-568 ofSEQ. ID. NO. 1 SEQ ID NO.: 18 “NBNint.antisence” Bottom Primer for NBNfrom human fetal brain cDNA  20 nt reverse complement to bases 633-652of SEQ. ID. NO. 1 SEQ ID NO.: 19 “NBNext.sence” Top Primer for wholehuman brain mRNA RT-PCR  17 nt complementary to bases 58-74 Of SEQ. ID.NO. 8. SEQ ID NO.: 20 “NBNext.antisence” Bottom Primer for whole humanbrain mRNA RT-PCR  16 nt reverse complement to bases 850-865 of SEQ. ID.NO. 8. SEQ ID NO.: 21 “NBNint.sence” NBN C2 Primer for screening mousefetal cDNA master plate  18 nt complementary to bases 1398-1415 of SEQ.ID. NO. 15. SEQ ID NO.: 22 “NBNint.antisence” NBN C2 as Primer forscreening mouse fetal cDNA master  20 nt plate. Reverse complement tobases 1598-1617 of SEQ. ID. NO. 15. SEQ ID NO.: 23 Primer Pair 1 SensePCR Primer for human genomic DNA amplification  29 nt complementary tobases 60-88 of SEQ. ID. NO. 3. SEQ ID NO.: 24 Primer Pair 1 AntisensePCR Primer for human genomic DNA amplification  27 nt Reverse complementto bases 835-861 of SEQ. ID. NO. 3. SEQ ID NO.: 25 Primer Pair 2 SensePCR Primer for human genomic DNA amplification  35 nt complementary tobases 1-35 of SEQ. ID. NO. 3. SEQ ID NO.: 26 Primer Pair 2 Antisense PCRPrimer for human genomic DNA amplification  34 nt reverse complement tobases 786-819 of SEQ. ID. NO. 3. SEQ ID NO.: 27 Antisense alkalinephosphatase conjugated hybridization probe,  30 nt complimentary tobases 1140-1169 of mouse neuroblastin cDNA. SEQ ID NO.: 28“NBNext.sence” Top Primer for whole human brain mRNA RT-PCR  16 ntcomplementary to bases 116 of SEQ. ID. NO. 1 SEQ ID NO.: 29 Syngene fromFIG. 14 of neublastin.  351 nt SEQ ID NO.: 30 Syngene from FIG. 15 ofHisneublastin.  414 nt SEQ ID NO.: 31 Primer for isolating neublastin. 39 nt SEQ ID NO.: 32 Primer for isolating neublastin.  39 nt SEQ IDNO.: 33 “NBNint.antisence” NBN primer; reverse complement to bases715-730  16 nt of SEQ. ID. NO. 8.

43 1 865 DNA Homo sapiens CDS (120)..(719) 5′UTR (1)..(119) 3′UTR(721)..(865) sig_peptide (120)..(179) mat_peptide (405)..(719)misc_structure (661)..(663) CARBOHYD Glycosylated Asparagine at Asn87 1ctaggagccc atgcccggcc tgatctcagc ccgaggacag cccctccttg aggtccttcc 60tccccaagcc cacctgggtg ccctctttct ccctgaggct ccacttggtc tctccgcgc 119 atgcct gcc ctg tgg ccc acc ctg gcc gct ctg gct ctg ctg agc agc 167 Met ProAla Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu Leu Ser Ser -95 -90 -85 -80gtc gca gag gcc tcc ctg ggc tcc gcg ccc cgc agc cct gcc ccc cgc 215 ValAla Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro Ala Pro Arg -75 -70 -65gaa ggc ccc ccg cct gtc ctg gcg tcc ccc gcc ggc cac ctg ccg ggg 263 GluGly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His Leu Pro Gly -60 -55 -50gga cgc acg gcc cgc tgg tgc agt gga aga gcc cgg cgg ccg cgc cgc 311 GlyArg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg Pro Arg Arg -45 -40 -35aga cac ttc tcg gcc cgc gcc ccc gcc gcc tgc acc ccc atc tgc tct 359 ArgHis Phe Ser Ala Arg Ala Pro Ala Ala Cys Thr Pro Ile Cys Ser -30 -25 -20tcc ccg cgg gtc cgc gcg gcg cgg ctg ggg ggc cgg gca gcg cgc tcg 407 SerPro Arg Val Arg Ala Ala Arg Leu Gly Gly Arg Ala Ala Arg Ser -15 -10 -5-1 1 ggc agc ggg ggc gcg ggg tgc cgc ctg cgc tcg cag ctg gtg ccg gtg 455Gly Ser Gly Gly Ala Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val 5 10 15cgc gcg ctc ggc ctg ggc cac cgc tcc gac gag ctg gtg cgt ttc cgc 503 ArgAla Leu Gly Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg 20 25 30 ttctgc acc ggc tcc tgc ccg cgc gcg cgc tct cca cac gac ctc agc 551 Phe CysThr Gly Ser Cys Pro Arg Ala Arg Ser Pro His Asp Leu Ser 35 40 45 ctg gccagc cta ctg ggc gcc ggg gcc ctg cga ccg ccc ccg ggc tcc 599 Leu Ala SerLeu Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser 50 55 60 65 cgg cccgtc agc cag ccc tgc tgc cga ccc acg cgc tac gaa gcg gtc 647 Arg Pro ValSer Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val 70 75 80 tcc ttc atggac gtc aac agc acc tgg aga acc gtg gac cgc ctc tcc 695 Ser Phe Met AspVal Asn Ser Thr Trp Arg Thr Val Asp Arg Leu Ser 85 90 95 gcc acc gcc tgcggc tgc ctg ggc tgagggctcg ctccagggct ttgcagactg 749 Ala Thr Ala Cys GlyCys Leu Gly 100 105 gacccttacc ggtggctctt cctgcctggg accctcccgcagagtcccac tagccagcgg 809 cctcagccag ggacgaaggc ctcaaagctg agaggcccctgccggtgggt gatgga 865 2 200 PRT Homo sapiens 2 Met Pro Ala Leu Trp ProThr Leu Ala Ala Leu Ala Leu Leu Ser Ser -95 -90 -85 -80 Val Ala Glu AlaSer Leu Gly Ser Ala Pro Arg Ser Pro Ala Pro Arg -75 -70 -65 Glu Gly ProPro Pro Val Leu Ala Ser Pro Ala Gly His Leu Pro Gly -60 -55 -50 Gly ArgThr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg Pro Arg Arg -45 -40 -35 ArgHis Phe Ser Ala Arg Ala Pro Ala Ala Cys Thr Pro Ile Cys Ser -30 -25 -20Ser Pro Arg Val Arg Ala Ala Arg Leu Gly Gly Arg Ala Ala Arg Ser -15 -10-5 -1 1 Gly Ser Gly Gly Ala Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val5 10 15 Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg20 25 30 Phe Cys Thr Gly Ser Cys Pro Arg Ala Arg Ser Pro His Asp Leu Ser35 40 45 Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser50 55 60 65 Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu AlaVal 70 75 80 Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val Asp Arg LeuSer 85 90 95 Ala Thr Ala Cys Gly Cys Leu Gly 100 105 3 861 DNA Homosapiens CDS (7)..(717) 5′UTR (1)..(6) 3′UTR (718)..(861) sig_peptide(7)..(174) mat_peptide (298)..(717) mat_peptide (370)..(717) mat_peptide(379)..(717) misc_structure (661)..(663) CARBOHYD glycosylatedAsparagine as Asn122 3 gagccc atg ccc ggc ctg atc tca gcc cga gga cagccc ctc ctt gag 48 Met Pro Gly Leu Ile Ser Ala Arg Gly Gln Pro Leu LeuGlu -95 -90 -85 gtc ctt cct ccc caa gcc cac ctg ggt gcc ctc ttt ctc cctgag gct 96 Val Leu Pro Pro Gln Ala His Leu Gly Ala Leu Phe Leu Pro GluAla -80 -75 -70 cca ctt ggt ctc tcc gcg cag cct gcc ctg tgg ccc acc ctggcc gct 144 Pro Leu Gly Leu Ser Ala Gln Pro Ala Leu Trp Pro Thr Leu AlaAla -65 -60 -55 ctg gct ctg ctg agc agc gtc gca gag gcc tcc ctg ggc tccgcg ccc 192 Leu Ala Leu Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser AlaPro -50 -45 -40 cgc agc cct gcc ccc cgc gaa ggc ccc ccg cct gtc ctg gcgtcc ccc 240 Arg Ser Pro Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala SerPro -35 -30 -25 -20 gcc ggc cac ctg ccg ggg gga cgc acg gcc cgc tgg tgcagt gga aga 288 Ala Gly His Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys SerGly Arg -15 -10 -5 gcc cgg cgg ccg ccg ccg cag cct tct cgg ccc gcg cccccg ccg cct 336 Ala Arg Arg Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro ProPro Pro -1 1 5 10 gca ccc cca tct gct ctt ccc cgc ggg ggc cgc gcg gcgcgg gct ggg 384 Ala Pro Pro Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala ArgAla Gly 15 20 25 ggc ccg ggc aac cgc gct cgg gca gcg ggg gcg cgg ggc tgccgc ctg 432 Gly Pro Gly Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys ArgLeu 30 35 40 45 cgc tcg cag ctg gtg ccg gtg cgc gcg ctc ggc ctg ggc caccgc tcc 480 Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His ArgSer 50 55 60 gac gag ctg gtg cgt ttc cgc ttc tgc agc ggc tcc tgc cgc cgcgcg 528 Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala65 70 75 cgc tct cca cac gac ctc agc ctg gcc agc cta ctg ggc gcc ggg gcc576 Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala 8085 90 ctg cga ccg ccc ccg ggc tcc cgg ccc gtc agc cag ccc tgc tgc cga624 Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg 95100 105 ccc acg cgc tac gaa gcg gtc tcc ttc atg gac gtc aac agc acc tgg672 Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp 110115 120 125 aga acc gtg gac cgc ctc tcc gcc aac ccc tgc ggc tgc ctg ggc717 Arg Thr Val Asp Arg Leu Ser Ala Asn Pro Cys Gly Cys Leu Gly 130 135140 tgagggctcg ctccagggct ttgcagactg gacccttacc ggtggctctt cctgcctggg777 accctcccgc agagtcccac tagccagcgg cctcagccag ggacgaaggc ctcaaagctg837 agaggcccct gccggtgggt gatg 861 4 237 PRT Homo sapiens 4 Met Pro GlyLeu Ile Ser Ala Arg Gly Gln Pro Leu Leu Glu Val Leu -95 -90 -85 Pro ProGln Ala His Leu Gly Ala Leu Phe Leu Pro Glu Ala Pro Leu -80 -75 -70 GlyLeu Ser Ala Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala -65 -60 -55-50 Leu Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser -45-40 -35 Pro Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly-30 -25 -20 His Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg AlaArg -15 -10 -5 Arg Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro ProAla Pro -1 1 5 10 15 Pro Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg AlaGly Gly Pro 20 25 30 Gly Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys ArgLeu Arg Ser 35 40 45 Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His ArgSer Asp Glu 50 55 60 Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg ArgAla Arg Ser 65 70 75 Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala GlyAla Leu Arg 80 85 90 95 Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro CysCys Arg Pro Thr 100 105 110 Arg Tyr Glu Ala Val Ser Phe Met Asp Val AsnSer Thr Trp Arg Thr 115 120 125 Val Asp Arg Leu Ser Ala Asn Pro Cys GlyCys Leu Gly 130 135 140 5 140 PRT Homo sapiens VARIANT (134) Wherein Xaaat position 134 designates Asn or Thr 5 Pro Pro Pro Gln Pro Ser Arg ProAla Pro Pro Pro Pro Ala Pro Pro 1 5 10 15 Ser Ala Leu Pro Arg Gly GlyArg Ala Ala Arg Ala Gly Gly Pro Gly 20 25 30 Asn Arg Ala Arg Ala Ala GlyAla Arg Gly Cys Arg Leu Arg Ser Gln 35 40 45 Leu Val Pro Val Arg Ala LeuGly Leu Gly His Arg Ser Asp Glu Leu 50 55 60 Val Arg Phe Arg Phe Cys SerGly Ser Cys Arg Arg Ala Arg Ser Pro 65 70 75 80 His Asp Leu Ser Leu AlaSer Leu Leu Gly Ala Gly Ala Leu Arg Pro 85 90 95 Pro Pro Gly Ser Arg ProVal Ser Gln Pro Cys Cys Arg Pro Thr Arg 100 105 110 Tyr Glu Ala Val SerPhe Met Asp Val Asn Ser Thr Trp Arg Thr Val 115 120 125 Asp Arg Leu SerAla Xaa Xaa Cys Gly Cys Leu Gly 130 135 140 6 116 PRT Homo sapiensVARIANT (110) Wherein Xaa at position 110 designates Asn or Thr 6 AlaAla Arg Ala Gly Gly Pro Gly Asn Arg Ala Arg Ala Ala Gly Ala 1 5 10 15Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly 20 25 30Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly 35 40 45Ser Cys Arg Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu 50 55 60Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser 65 70 7580 Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp 85 9095 Val Asn Ser Thr Trp Arg Thr Val Asp Arg Leu Ser Ala Xaa Xaa Cys 100105 110 Gly Cys Leu Gly 115 7 113 PRT Homo sapiens VARIANT (107) WhereinXaa at position 107 designates Asn or Thr 7 Ala Gly Gly Pro Gly Asn ArgAla Arg Ala Ala Gly Ala Arg Gly Cys 1 5 10 15 Arg Leu Arg Ser Gln LeuVal Pro Val Arg Ala Leu Gly Leu Gly His 20 25 30 Arg Ser Asp Glu Leu ValArg Phe Arg Phe Cys Ser Gly Ser Cys Arg 35 40 45 Arg Ala Arg Ser Pro HisAsp Leu Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60 Gly Ala Leu Arg Pro ProPro Gly Ser Arg Pro Val Ser Gln Pro Cys 65 70 75 80 Cys Arg Pro Thr ArgTyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95 Thr Trp Arg Thr ValAsp Arg Leu Ser Ala Xaa Xaa Cys Gly Cys Leu 100 105 110 Gly 8 861 DNAHomo sapiens CDS (58)..(717) 5′UTR (1)..(57) 3′UTR (718)..(861)sig_peptide (58)..(174) mat_peptide (298)..(717) mat_peptide(370)..(717) mat_peptide (379)..(717) misc_structure (661)..(663)CARBOHYD glycosylated asparagine at Asn122 8 aggagggtgg gggaacagctcaacaatggc tgatgggcgc tcctggtgtt gatagag 57 atg gaa ctt gga ctt gga ggcctc tcc acg ctg tcc cac tgc ccc tgg 105 Met Glu Leu Gly Leu Gly Gly LeuSer Thr Leu Ser His Cys Pro Trp -80 -75 -70 -65 cct agg cgg cag cct gccctg tgg ccc acc ctg gcc gct ctg gct ctg 153 Pro Arg Arg Gln Pro Ala LeuTrp Pro Thr Leu Ala Ala Leu Ala Leu -60 -55 -50 ctg agc agc gtc gca gaggcc tcc ctg ggc tcc gcg ccc cgc agc cct 201 Leu Ser Ser Val Ala Glu AlaSer Leu Gly Ser Ala Pro Arg Ser Pro -45 -40 -35 gcc ccc cgc gaa ggc cccccg cct gtc ctg gcg tcc ccc gcc ggc cac 249 Ala Pro Arg Glu Gly Pro ProPro Val Leu Ala Ser Pro Ala Gly His -30 -25 -20 ctg ccg ggg gga cgc acggcc cgc tgg tgc agt gga aga gcc cgg cgg 297 Leu Pro Gly Gly Arg Thr AlaArg Trp Cys Ser Gly Arg Ala Arg Arg -15 -10 -5 -1 ccg ccg ccg cag ccttct cgg ccc gcg ccc ccg ccg cct gca ccc cca 345 Pro Pro Pro Gln Pro SerArg Pro Ala Pro Pro Pro Pro Ala Pro Pro 1 5 10 15 tct gct ctt ccc cgcggg ggc cgc gcg gcg cgg gct ggg ggc ccg ggc 393 Ser Ala Leu Pro Arg GlyGly Arg Ala Ala Arg Ala Gly Gly Pro Gly 20 25 30 agc cgc gct cgg gca gcgggg gcg cgg ggc tgc cgc ctg cgc tcg cag 441 Ser Arg Ala Arg Ala Ala GlyAla Arg Gly Cys Arg Leu Arg Ser Gln 35 40 45 ctg gtg ccg gtg cgc gcg ctcggc ctg ggc cac cgc tcc gac gag ctg 489 Leu Val Pro Val Arg Ala Leu GlyLeu Gly His Arg Ser Asp Glu Leu 50 55 60 gtg cgt ttc cgc ttc tgc agc ggctcc tgc cgc cgc gcg cgc tct cca 537 Val Arg Phe Arg Phe Cys Ser Gly SerCys Arg Arg Ala Arg Ser Pro 65 70 75 80 cac gac ctc agc ctg gcc agc ctactg ggc gcc ggg gcc ctg cga ccg 585 His Asp Leu Ser Leu Ala Ser Leu LeuGly Ala Gly Ala Leu Arg Pro 85 90 95 ccc ccg ggc tcc cgg ccc gtc agc cagccc tgc tgc cga ccc acg cgc 633 Pro Pro Gly Ser Arg Pro Val Ser Gln ProCys Cys Arg Pro Thr Arg 100 105 110 tac gaa gcg gtc tcc ttc atg gac gtcaac agc acc tgg aga acc gtg 681 Tyr Glu Ala Val Ser Phe Met Asp Val AsnSer Thr Trp Arg Thr Val 115 120 125 gac cgc ctc tcc gcc acc gcc tgc ggctgc ctg ggc tgagggctcg 727 Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys LeuGly 130 135 140 ctccagggct ttgcagactg gacccttacc ggtggctctt cctgcctgggaccctcccgc 787 agagtcccac tagccagcgg cctcagccag ggacgaaggc ctcaaagctgagaggcccct 847 accggtgggt gatg 861 9 220 PRT Homo sapiens 9 Met Glu LeuGly Leu Gly Gly Leu Ser Thr Leu Ser His Cys Pro Trp -80 -75 -70 -65 ProArg Arg Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu -60 -55 -50Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro -45 -40-35 Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His -30-25 -20 Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg-15 -10 -5 -1 Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro AlaPro Pro 1 5 10 15 Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala GlyGly Pro Gly 20 25 30 Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg LeuArg Ser Gln 35 40 45 Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg SerAsp Glu Leu 50 55 60 Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg AlaArg Ser Pro 65 70 75 80 His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala GlyAla Leu Arg Pro 85 90 95 Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys CysArg Pro Thr Arg 100 105 110 Tyr Glu Ala Val Ser Phe Met Asp Val Asn SerThr Trp Arg Thr Val 115 120 125 Asp Arg Leu Ser Ala Thr Ala Cys Gly CysLeu Gly 130 135 140 10 140 PRT Homo sapiens CARBOHYD (122) glycosylatedasparagine 10 Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro AlaPro Pro 1 5 10 15 Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala GlyGly Pro Gly 20 25 30 Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg LeuArg Ser Gln 35 40 45 Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg SerAsp Glu Leu 50 55 60 Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg AlaArg Ser Pro 65 70 75 80 His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala GlyAla Leu Arg Pro 85 90 95 Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys CysArg Pro Thr Arg 100 105 110 Tyr Glu Ala Val Ser Phe Met Asp Val Asn SerThr Trp Arg Thr Val 115 120 125 Asp Arg Leu Ser Ala Thr Ala Cys Gly CysLeu Gly 130 135 140 11 116 PRT Homo sapiens CARBOHYD (98) glycosylatedasparagine 11 Ala Ala Arg Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala AlaGly Ala 1 5 10 15 Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val ArgAla Leu Gly 20 25 30 Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg PheCys Ser Gly 35 40 45 Ser Cys Arg Arg Ala Arg Ser Pro His Asp Leu Ser LeuAla Ser Leu 50 55 60 Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser ArgPro Val Ser 65 70 75 80 Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala ValSer Phe Met Asp 85 90 95 Val Asn Ser Thr Trp Arg Thr Val Asp Arg Leu SerAla Thr Ala Cys 100 105 110 Gly Cys Leu Gly 115 12 113 PRT Homo sapiensCARBOHYD (95) glycosylated asparagine 12 Ala Gly Gly Pro Gly Ser Arg AlaArg Ala Ala Gly Ala Arg Gly Cys 1 5 10 15 Arg Leu Arg Ser Gln Leu ValPro Val Arg Ala Leu Gly Leu Gly His 20 25 30 Arg Ser Asp Glu Leu Val ArgPhe Arg Phe Cys Ser Gly Ser Cys Arg 35 40 45 Arg Ala Arg Ser Pro His AspLeu Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60 Gly Ala Leu Arg Pro Pro ProGly Ser Arg Pro Val Ser Gln Pro Cys 65 70 75 80 Cys Arg Pro Thr Arg TyrGlu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95 Thr Trp Arg Thr Val AspArg Leu Ser Ala Thr Ala Cys Gly Cys Leu 100 105 110 Gly 13 102 DNA Homosapiens 13 cctggccagc ctactgggcg ccggggccct gcgaccgccc ccgggctcccggcccgtcag 60 ccagccctgc tgccgaccca cgcgctacga agcggtctcc tt 102 14 220DNA Murinae gen. sp. 14 ggccaccgct ccgacgagct gatacgtttc cgcttctgcagcggctcgtg ccgccgagca 60 cgctcccagc acgatctcag tctggccagc ctactgggcgctggggccct acggtcgcct 120 cccgggtccc ggccgatcag ccagccctgc tgccggcccactcgctatga ggccgtctcc 180 ttcatggacg tgaacagcac ctggagaacc gtggaccgcc220 15 2136 DNA Murinae gen. sp. CDS (975)..(1646) 15 gcggccgcgaattcggcacg agggcgtctc gctgcagccc gcgatctcta ctctgcctcc 60 tggggtcttctccaaatgtc tagcccccac ctagagggac ctagcctagc cagcggggac 120 cggatccggagggtggagcg gccaggtgag ccctgaaagg tggggcgggg cgggggcgct 180 ctgggccccaccccgggatc tggtgacgcc ggggctggaa tttgacaccg gacggcggcg 240 ggcaggaggctgctgaggga tggagttggg ctcggccccc agatgcggcc cgcgggctct 300 gccagcaacaagtccctcgg gccccagccc tcgctgcgac tggggcttgg agccctgcac 360 ccaagggcacagaccggctg ccaaggcccc acttttaact aaaagaggcg ctgccaggtg 420 cacaactctgggcatgatcc acttgagctt cgggggaaag cccagcactg gtcccaggag 480 aggcgcctagaaggacacgg accaggaccc ctttggtatg gagtgaacgc tgagcatgga 540 gtggaaggaactcaagttac tactttctcc aaccaccctg gtaccttcag ccctgaagta 600 cagagcagaagggtcttaga agacaggacc acagctgtgt gagtctcccc cctgaggcct 660 tagacgatctctgagctcag ctgagctttg tttgcccatc tggagaagtg agccattgat 720 tgaccttgtggcatcgcgaa ggaacaggtc ctgccaagca cctaacacag agagcaaggt 780 tctccatcgcagctaccgct gctgagttga ctctagctac tccaacctcc tgggtcgctt 840 cgagagactggagtggaagg aggaataccc caaaggataa ctaactcatc tttcagtttg 900 caagctgccgcaggaagagg gtggggaaac gggtccacga aggcttctga tgggagcttc 960 tggagccgaaagct atg gaa ctg gga ctt gca gag cct act gca ttg tcc 1010 Met Glu LeuGly Leu Ala Glu Pro Thr Ala Leu Ser 1 5 10 cac tgc ctc cgg cct agg tggcag tca gcc tgg tgg cca acc cta gct 1058 His Cys Leu Arg Pro Arg Trp GlnSer Ala Trp Trp Pro Thr Leu Ala 15 20 25 gtt cta gcc ctg ctg agc tgc gtcaca gaa gct tcc ctg gac cca atg 1106 Val Leu Ala Leu Leu Ser Cys Val ThrGlu Ala Ser Leu Asp Pro Met 30 35 40 tcc cgc agc ccc gcc gct cgc gac ggtccc tca ccg gtc ttg gcg ccc 1154 Ser Arg Ser Pro Ala Ala Arg Asp Gly ProSer Pro Val Leu Ala Pro 45 50 55 60 ccc acg gac cac ctg cct ggg gga cacact gcg cat ttg tgc agc gaa 1202 Pro Thr Asp His Leu Pro Gly Gly His ThrAla His Leu Cys Ser Glu 65 70 75 aga acc ctg cga ccc ccg cct cag tct cctcag ccc gca ccc ccg ccg 1250 Arg Thr Leu Arg Pro Pro Pro Gln Ser Pro GlnPro Ala Pro Pro Pro 80 85 90 cct ggt ccc gcg ctc cag tct cct ccc gct gcgctc cgc ggg gca cgc 1298 Pro Gly Pro Ala Leu Gln Ser Pro Pro Ala Ala LeuArg Gly Ala Arg 95 100 105 gcg gcg cgt gca gga acc cgg agc agc cgc gcacgg acc aca gat gcg 1346 Ala Ala Arg Ala Gly Thr Arg Ser Ser Arg Ala ArgThr Thr Asp Ala 110 115 120 cgc ggc tgc cgc ctg cgc tcg cag ctg gtg ccggtg agc gcg ctc ggc 1394 Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro ValSer Ala Leu Gly 125 130 135 140 cta ggc cac agc tcc gac gag ctg ata cgtttc cgc ttc tgc agc ggc 1442 Leu Gly His Ser Ser Asp Glu Leu Ile Arg PheArg Phe Cys Ser Gly 145 150 155 tcg tgc cgc cga gca cgc tcc cag cac gatctc agt ctg gcc agc cta 1490 Ser Cys Arg Arg Ala Arg Ser Gln His Asp LeuSer Leu Ala Ser Leu 160 165 170 ctg ggc gct ggg gcc cta cgg tcg cct cccggg tcc cgg ccg atc agc 1538 Leu Gly Ala Gly Ala Leu Arg Ser Pro Pro GlySer Arg Pro Ile Ser 175 180 185 cag ccc tgc tgc cgg ccc act cgc tat gaggcc gtc tcc ttc atg gac 1586 Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu AlaVal Ser Phe Met Asp 190 195 200 gtg aac agc acc tgg agg acc gtg gac cacctc tcc gcc act gcc tgc 1634 Val Asn Ser Thr Trp Arg Thr Val Asp His LeuSer Ala Thr Ala Cys 205 210 215 220 ggc tgt ctg ggc tgaggatgatctatctccaa gcctttgcac actagaccca 1686 Gly Cys Leu Gly tgtgttgccctacctggaac agctccaccg ggcctcacta accaggagcc tcaactcagc 1746 aggatatggaggctgcagag ctcaggcccc aggccggtga gtgacagacg tcgtcggcat 1806 gacagacagagtgaaagatg tcggaaccac tgaccaacag tcccaagttg ttcatggatc 1866 ccagctctacagacaggaga aacctcagct aaagagaact cctctgggag aatccagaaa 1926 tggccctctgtcctggggaa tgaattttga agagatatat atacatatat acattgtagt 1986 cgcgttgctggaccagcctg tgctgaaacc agtcccgtgt tcacttgtgg aagccgaagc 2046 cctatttattatttctaaat tatttattta ctttgaaaaa aaacggccaa gtcggcctcc 2106 ctttagtgagggttaatttg tgatcccggg 2136 16 224 PRT Murinae gen. sp. 16 Met Glu LeuGly Leu Ala Glu Pro Thr Ala Leu Ser His Cys Leu Arg 1 5 10 15 Pro ArgTrp Gln Ser Ala Trp Trp Pro Thr Leu Ala Val Leu Ala Leu 20 25 30 Leu SerCys Val Thr Glu Ala Ser Leu Asp Pro Met Ser Arg Ser Pro 35 40 45 Ala AlaArg Asp Gly Pro Ser Pro Val Leu Ala Pro Pro Thr Asp His 50 55 60 Leu ProGly Gly His Thr Ala His Leu Cys Ser Glu Arg Thr Leu Arg 65 70 75 80 ProPro Pro Gln Ser Pro Gln Pro Ala Pro Pro Pro Pro Gly Pro Ala 85 90 95 LeuGln Ser Pro Pro Ala Ala Leu Arg Gly Ala Arg Ala Ala Arg Ala 100 105 110Gly Thr Arg Ser Ser Arg Ala Arg Thr Thr Asp Ala Arg Gly Cys Arg 115 120125 Leu Arg Ser Gln Leu Val Pro Val Ser Ala Leu Gly Leu Gly His Ser 130135 140 Ser Asp Glu Leu Ile Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg145 150 155 160 Ala Arg Ser Gln His Asp Leu Ser Leu Ala Ser Leu Leu GlyAla Gly 165 170 175 Ala Leu Arg Ser Pro Pro Gly Ser Arg Pro Ile Ser GlnPro Cys Cys 180 185 190 Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met AspVal Asn Ser Thr 195 200 205 Trp Arg Thr Val Asp His Leu Ser Ala Thr AlaCys Gly Cys Leu Gly 210 215 220 17 18 DNA Artificial SequenceDescription of Artificial Sequence PCR Primer 17 cctggccagc ctactggg 1818 20 DNA Artificial Sequence Description of Artificial Sequence PCRPrimer 18 aaggagaccg cttcgtagcg 20 19 17 DNA Artificial SequenceDescription of Artificial Sequence PCR Primer 19 atggaacttg gacttgg 1720 16 DNA Artificial Sequence Description of Artificial Sequence PCRPrimer 20 tccatcaccc accggc 16 21 18 DNA Artificial Sequence Descriptionof Artificial Sequence PCR Primer 21 ggccaccgct ccgacgag 18 22 20 DNAArtificial Sequence Description of Artificial Sequence PCR Primer 22ggcggtccac ggttctccag 20 23 29 DNA Artificial Sequence Description ofArtificial Sequence PCR Primer 23 ccaagcccac ctgggtgccc tctttctcc 29 2427 DNA Artificial Sequence Description of Artificial Sequence PCR Primer24 catcacccac cggcaggggc ctctcag 27 25 35 DNA Artificial SequenceDescription of Artificial Sequence PCR Primer 25 gagcccatgc ccggcctgatctcagcccga ggaca 35 26 34 DNA Artificial Sequence Description ofArtificial Sequence PCR Primer 26 ccctggctga ggccgctggc tagtgggact ctgc34 27 31 DNA Artificial Sequence Description of Artificial SequenceHybridization Probe 27 ncaggtggtc cgtggggggc gccaagaccg g 31 28 16 DNAArtificial Sequence Description of Artificial Sequence PCR primer 28ctaggagccc atgccc 16 29 351 DNA Homo sapiens 29 atggctggag gaccgggatctcgtgctcgt gcagcaggag cacgtggctg tcgtctgcgt 60 tctcaactag tgccggtgcgtgcactcgga ctgggacacc gttccgacga actagtacgt 120 tttcgttttt gttcaggatcttgtcgtcgt gcacgttctc cgcatgatct atctctagca 180 tctctactag gagccggagcactaagaccg ccgccgggat ctagacctgt atctcaacct 240 tgttgtagac ctactagatacgaagcagta tctttcatgg acgtaaactc tacatggaga 300 accgtagata gactatctgcaaccgcatgt ggctgtctag gatgataata g 351 30 414 DNA Homo sapiens 30atgggccatc atcatcatca tcatcatcat catcactcga gcggccatat cgacgacgac 60gacaaggctg gaggaccggg atctcgtgct cgtgcagcag gagcacgtgg ctgtcgtctg 120cgttctcaac tagtgccggt gcgtgcactc ggactgggac accgttccga cgaactagta 180cgttttcgtt tttgttcagg atcttgtcgt cgtgcacgtt ctccgcatga tctatctcta 240gcatctctac taggagccgg agcactaaga ccgccgccgg gatctagacc tgtatctcaa 300ccttgttgta gacctactag atacgaagca gtatctttca tggacgtaaa ctctacatgg 360agaaccgtag atagactatc tgcaaccgca tgtggctgtc taggatgata atag 414 31 39DNA Artificial Sequence Description of Artificial Sequence PCR primer 31aaggaaaaaa gcggccgcca tggaacttgg acttggagg 39 32 39 DNA ArtificialSequence Description of Artificial Sequence PCR primer 32 ttttttccttggcggccgct cagcccaggc agccgcagg 39 33 16 DNA Artificial SequenceDescription of Artificial Sequence primer 33 gagcgagccc tcagcc 16 34 197PRT Homo sapiens 34 Met Gln Arg Trp Lys Ala Ala Ala Leu Ala Ser Val LeuCys Ser Ser 1 5 10 15 Val Leu Ser Ile Trp Met Cys Arg Glu Gly Leu LeuLeu Ser His Arg 20 25 30 Leu Gly Pro Ala Leu Val Pro Leu His Arg Leu ProArg Thr Leu Asp 35 40 45 Ala Arg Ile Ala Arg Leu Ala Gln Tyr Arg Ala LeuLeu Gln Gly Ala 50 55 60 Pro Asp Ala Met Glu Leu Arg Glu Leu Thr Pro TrpAla Gly Arg Pro 65 70 75 80 Pro Gly Pro Arg Arg Arg Ala Gly Pro Arg ArgArg Arg Ala Arg Ala 85 90 95 Arg Leu Gly Ala Arg Pro Cys Gly Leu Arg GluLeu Glu Val Arg Val 100 105 110 Ser Glu Leu Gly Leu Gly Tyr Ala Ser AspGlu Thr Val Leu Phe Arg 115 120 125 Tyr Cys Ala Gly Ala Cys Glu Ala AlaAla Arg Val Tyr Asp Leu Gly 130 135 140 Leu Arg Arg Leu Arg Gln Arg ArgArg Leu Arg Arg Glu Arg Val Arg 145 150 155 160 Ala Gln Pro Cys Cys ArgPro Thr Ala Tyr Glu Asp Glu Val Ser Phe 165 170 175 Leu Asp Ala His SerArg Tyr His Thr Val His Glu Leu Ser Ala Arg 180 185 190 Glu Cys Ala CysVal 195 35 220 PRT Homo sapiens 35 Met Glu Leu Gly Leu Gly Gly Leu SerThr Leu Ser His Cys Pro Trp 1 5 10 15 Pro Arg Arg Gln Pro Ala Leu TrpPro Thr Leu Ala Ala Leu Ala Leu 20 25 30 Leu Ser Ser Val Ala Glu Ala SerLeu Gly Ser Ala Pro Arg Ser Pro 35 40 45 Ala Pro Arg Glu Gly Pro Pro ProVal Leu Ala Ser Pro Ala Gly His 50 55 60 Leu Pro Gly Gly Arg Thr Ala ArgTrp Cys Ser Gly Arg Ala Arg Arg 65 70 75 80 Pro Pro Pro Gln Pro Ser ArgPro Ala Pro Pro Pro Pro Ala Pro Pro 85 90 95 Ser Ala Leu Pro Arg Gly GlyArg Ala Ala Arg Ala Gly Gly Pro Gly 100 105 110 Ser Arg Ala Arg Ala AlaGly Ala Arg Gly Cys Arg Leu Arg Ser Gln 115 120 125 Leu Val Pro Val ArgAla Leu Gly Leu Gly His Arg Ser Asp Glu Leu 130 135 140 Val Arg Phe ArgPhe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser Pro 145 150 155 160 His AspLeu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro 165 170 175 ProPro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg 180 185 190Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val 195 200205 Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 210 215 220 36 156PRT Homo sapiens 36 Met Ala Val Gly Lys Phe Leu Leu Gly Ser Leu Leu LeuLeu Ser Leu 1 5 10 15 Gln Leu Gly Gln Gly Trp Gly Pro Asp Ala Arg GlyVal Pro Val Ala 20 25 30 Asp Gly Glu Phe Ser Ser Glu Gln Val Ala Lys AlaGly Gly Thr Trp 35 40 45 Leu Gly Thr His Arg Pro Leu Ala Arg Leu Arg ArgAla Leu Ser Gly 50 55 60 Pro Cys Gln Leu Trp Ser Leu Thr Leu Ser Val AlaGlu Leu Gly Leu 65 70 75 80 Gly Tyr Ala Ser Glu Glu Lys Val Ile Phe ArgTyr Cys Ala Gly Ser 85 90 95 Cys Pro Arg Gly Ala Arg Thr Gln His Gly LeuAla Leu Ala Arg Leu 100 105 110 Gln Gly Gln Gly Arg Ala His Gly Gly ProCys Cys Arg Pro Thr Arg 115 120 125 Tyr Thr Asp Val Ala Phe Leu Asp AspArg His Arg Trp Gln Arg Leu 130 135 140 Pro Gln Leu Ser Ala Ala Ala CysGly Cys Gly Gly 145 150 155 37 211 PRT Homo sapiens 37 Met Lys Leu TrpAsp Val Val Ala Val Cys Leu Val Leu Leu His Thr 1 5 10 15 Ala Ser AlaPhe Pro Leu Pro Ala Gly Lys Arg Pro Pro Glu Ala Pro 20 25 30 Ala Glu AspArg Ser Leu Gly Arg Arg Arg Ala Pro Phe Ala Leu Ser 35 40 45 Ser Asp SerAsn Met Pro Glu Asp Tyr Pro Asp Gln Phe Asp Asp Val 50 55 60 Met Asp PheIle Gln Ala Thr Ile Lys Arg Leu Lys Arg Ser Pro Asp 65 70 75 80 Lys GlnMet Ala Val Leu Pro Arg Arg Glu Arg Asn Arg Gln Ala Ala 85 90 95 Ala AlaAsn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg 100 105 110 GlyLys Asn Arg Gly Cys Val Leu Thr Ala Ile His Leu Asn Val Thr 115 120 125Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu Ile Phe Arg Tyr 130 135140 Cys Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr Asp Lys Ile Leu 145150 155 160 Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Ser Asp Lys Val GlyGln 165 170 175 Ala Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu Ser PheLeu Asp 180 185 190 Asp Asn Leu Val Tyr His Ile Leu Arg Lys His Ser AlaLys Arg Cys 195 200 205 Gly Cys Ile 210 38 18 DNA Artificial SequenceDescription of Artificial Sequence primer 38 gctggcccgg ctgcaggg 18 3918 DNA Artificial Sequence Description of Artificial Sequence primer 39gctgcgacga ctgcgcca 18 40 18 DNA Artificial Sequence Description ofArtificial Sequence primer 40 attgaaaaac ttatccag 18 41 20 DNAArtificial Sequence Description of Artificial Sequence primer 41taggccacgt cggtgtagcg 20 42 23 DNA Artificial Sequence Description ofArtificial Sequence primer 42 aaggacacct cgtcctcgta ggc 23 43 23 DNAArtificial Sequence Description of Artificial Sequence primer 43aacgacaggt catcatcaaa ggc 23

We claim:
 1. An isolated neublastin polypeptide with neurotrophic activity comprising the following: (a) seven conserved cysteine residues at positions 8, 35, 39, 72, 73, 101, and 103 when numbered in accordance with SEQ ID NO. 2; (b) amino acid residues as follows: C at position 8, L at position 10, V at position 17, L at position 20, G at position 21, L at position 22, G at position 23, E at position 28, F at position 32, R at position 33, F at position 34, C at position 35, G at position 37, C at position 39, C at position 72, C at position 73, R at position 74, P at position 75, F at position 83, D at position 85, S at position 97, A at position 98, C at position 101 and C at position 103, each when numbered in accordance with SEQ ID NO. 2; (c) an LGLG repeat, an FRFC motif, a QPCCRP motif, and a SATACGC motif; and (d) an amino acid sequence comprising at least 90% sequence identity to AA₁-AA₁₀₅ of SEQ ID NO.
 2. 2. The polypeptide of claim 1, wherein said polypeptide is coded for by a nucleic acid selected from the group consisting of: a) nucleotides 405-719 of SEQ ID NO. 1; and b) a nucleotide sequence that hybridises specifically to a complement sequence of nucleotides 405-719 of SEQ ID NO. 1 under high stringency solution hybridization conditions where the hybridization conditions comprise pre-soaking of a filter containing the sequence of nucleotides in 5×Sodium chloride/Sodium citrate (SSC) for 10 minutes, pre-hybridization of the filter in a solution of 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA, hybridization in the same solution containing a concentration of 10 ng/ml of a random-primed ³²P-dCTP-labeled (specific activity>1×10⁹ cpm/μg) probe for 12 hours at approximately 45° C. and washing the filter for thirty minutes in 0.1×SSC, 0.5% SDS at a temperature of at least 70° C.; wherein the nucleotide sequence codes on-expression for a neublastin polypeptide comprising all of the following characteristics: (i) seven conserved cysteine residues at positions 8, 35, 39, 72, 73, 101, and 103 when numbered in accordance with SEQ ID NO. 2; (ii) amino acid residues as follows: C at position 8, L at position 10, V at position 17, L at position 20, G at position 21, L at position 22, G at position 23, E at position 28, F at position 32, R at position 33, F at position 34, C at position 35, G at position 37, C at position 39, C at position 72, C at position 73, R at position 74, P at position 75, F at position 83, D at position 85, S at position 97, A at position 98, C at position 101 and C at position 103, each when numbered in accordance with SEQ ID NO. 2; (iii) an LGLG repeat, an FRFC motif, a QPCCRP motif, and a SATACGC motif; and (iv) an amino acid sequence having at least 90% sequence identity with AA₁-AA105 of SEQ ID NO.
 2. 3. The neublastin polypeptide of claim 1, wherein the polypeptide has a C-terminal amino acid sequence as set forth in AA₇₂-AA₁₀₅ of SEQ. ID. NO.
 2. 4. The neublastin polypeptide of claim 1 wherein the polypeptide has a C-terminal amino acid sequence as set forth in AA₄₁-AA₁₀₅ of SEQ. ID. NO.
 2. 5. The neublastin polypeptide of claim 1 comprising conservative amino acid substitutions, wherein the conservative amino acid substitutions represent less than 10% of the total number of residues in the polypeptide.
 6. The neublastin polypeptide of claim 5 wherein the conservative amino acid substitutions represent less than 2% of the polypeptide.
 7. The neublastin polypeptide of claim 5 wherein the conservative amino acid substitutions represent a single amino acid substitution in the mature sequence, wherein both the substituted and replacement amino acids are non-cyclic.
 8. The polypeptide according to any one of claims 1-7 wherein said polypeptide is glycosylated.
 9. A composition comprising the polypeptide according to any one of claims 1-8 and a pharmaceutically acceptable carrier. 